Nucleic acid encoding mammalian mu opioid receptor

ABSTRACT

The invention relates generally to compositions of and methods for obtaining mu opioid receptor polypeptides. The invention relates as well to polynucleotides encoding mu opioid receptor polypeptides, the recombinant vectors carrying those sequences, the recombinant host cells including either the sequences or vectors, and recombinant opioid receptor polypeptides. The invention includes as well, methods for using the isolated, recombinant receptor polypeptide in assays designed to select and improve substances capable of interacting with mu opioid receptor polypeptides for use in diagnostic, drug design and therapeutic applications.

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 08/056,886, filed Mar. 8, 1993, now abandoned.

FIELD OF THE INVENTION

This invention relates generally to compositions of and methods forobtaining mu opioid receptors. The invention relates as well to the DNAsequences encoding mu opioid receptors, the recombinant vectors carryingthose sequences, the recombinant host cells including either thesequences or vectors, and recombinant mu opioid receptor polypeptides.The invention includes as well methods for using the isolated,recombinant receptor polypeptides in assays designed to select andimprove among candidate substances such as agonists and antagonists ofmu opioid receptors and polypeptides for use in diagnostic, drug designand therapeutic applications.

BACKGROUND OF THE INVENTION

Opioid drugs have various effects on perception of pain, consciousness,motor control, mood, and autonomic function and can also induce physicaldependence (Koob et al., 1992). The endogenous opioid system plays animportant role in modulating endocrine, cardiovascular, respiratory,gastrointestinal and immune functions (Olson et al., 1989). Opioidsexert their actions by binding to specific membrane-associated receptorslocated throughout the central and peripheral nervous system (Pert etal., 1973). The endogenous ligands of these opioid receptors have beenidentified as a family of more than 20 opioid peptides that derive fromthe three precursor proteins proopiomelanocortin, proenkephalin, andprodynorphin (Hughes et al., 1975; Akil et al., 1984). Although theopioid peptides belong to a class of molecules distinct from the opioidalkaloids, they share common structural features including a positivecharge juxtaposed with an aromatic ring that is required for interactionwith the receptor (Bradbury et al., 1976).

Pharmacological studies have suggested that there are numerous classesof opioid receptors, including those designated δ, κ, and μ (Simon,1991; Lutz et al., 1992). The classes differ in their affinity forvarious opioid ligands and in their cellular distribution. The differentclasses of opioid receptors are believed to serve differentphysiological functions (Olson et al., 1989; Simon, 1991; Lutz andPfister, 1992). However, there is substantial overlap of function aswell as of distribution. Biochemical characterization of opioidreceptors from many groups reports a molecular mass of ≈60,000 Da forall three subtypes, suggesting that they could be related molecules (Lohet al., 1990). Moreover, the similarity between the three receptorsubtypes is supported by the isolation of (i) anti-idiotypic monoclonalantibodies competing with both μ and δ ligands but not competing with κligands (Gramsch et al., 1988; Coscia et al., 1991) and (ii) amonoclonal antibody raised against the purified μ receptor thatinteracts with both μ and κ receptors (Bero et al., 1988).

Morphine interacts principally with μ receptors and peripheraladministration of this opioid induces release of enkephalins (Bertolucciet al., 1992). The δ receptors bind with the greatest affinity toenkephalins and have a more discrete distribution in the brain thaneither μ or κ receptors, with high concentrations in the basal gangliaand limbic regions. Thus, enkephalins may mediate part of thephysiological response to morphine, presumably by interacting with δreceptors. Despite pharmacological and physiological heterogeneity, atleast some types of opioid receptors inhibit adenylate cyclase, increaseK⁺ conductance, and inactivate Ca²⁺ channels through a pertussistoxin-sensitive mechanism (Puttfarcken et al., 1988; Attali et al.,1989; Hsia et al., 1984). These results and others suggest that opioidreceptors belong to the large family of cell surface receptors thatsignal through G proteins (Di Chiara et al., 1992; Loh et al., 1990).

Several attempts to clone cDNAs encoding opioid receptors have beenreported. A cDNA encoding an opioid-binding protein (OBCAM) with μselectivity was isolated (Schofield et al., 1989), but the predictedprotein lacks transmembrane domains, presumed necessary for signaltransduction. More recently, the isolation of another cDNA was reported,which was obtained by expression cloning (Xie et al., 1992). The deducedprotein sequence displays seven putative transmembrane domains and isvery similar to the human neuromedin K receptor. However, the affinityof opioid ligands for this receptor expressed in COS cells is two ordersof magnitude below the expected value, and no subtype selectivity can beshown

Many cell surface receptor/transmembrane systems consist of at leastthree membrane-bound polypeptide components: (a) a cell-surfacereceptor; (b) an effector, such as an ion channel or the enzymeadenylate cyclase; and (c) a guanine nucleotide-binding regulatorypolypeptide or G protein, that is coupled to both the receptor and itseffector.

G protein-coupled receptors mediate the actions of extracellular signalsas diverse as light, odorants, peptide hormones and neurotransmitters.Such receptors have been identified in organisms as evolutionarilydivergent as yeast and man. Nearly all G protein-coupled receptors bearsequence similarities with one another, and it is thought that all sharea similar topological motif consisting of seven hydrophobic (andpotentially α-helical) segments that span the lipid bilayer (Dohlman etal., 1987; Dohlman et al., 1991).

G proteins consist of three tightly associated subunits, α, β and γ(1:1:1) in order of decreasing mass. Following agonist binding to thereceptor, a conformational change is transmitted to the G protein, whichcauses the Gα-subunit to exchange a bound GDP for GTP and to dissociatefrom the βγ-subunits. The GTP-bound form of the α-subunit is typicallythe effector-modulating moiety. Signal amplification results from theability of a single receptor to activate many G protein molecules, andfrom the stimulation by Gα-GTP of many catalytic cycles of the effector.

The family of regulatory G proteins comprises a multiplicity ofdifferent α-subunits (greater than twenty in man), which associate witha smaller pool of β- and γ-subunits (greater than four each) (Strothmanand Simon, 1991). Thus, it is anticipated that differences in theα-subunits probably distinguish the various G protein oligomers,although the targeting or function of the various α-subunits might alsodepend on the βγ subunits with which they associate (Strothman andSimon, 1991).

Improvements in cell culture and in pharmacological methods, and morerecently, use of molecular cloning and gene expression techniques haveled to the identification and characterization of manyseven-transmembrane segment receptors, including new sub-types andsub-sub-types of previously identified receptors. The α₁ andα₂-adrenergic receptors once thought to each consist of single receptorspecies, are now known to each be encoded by at least three distinctgenes (Kobilka et al., 1987; Regan et al., 1988; Cotecchia et al., 1988;Lomasney, 1990). In addition to rhodopsin in rod cells, which mediatesvision in dim light, three highly similar cone pigments mediating colorvision have been cloned (Nathans et al., 1986A; and Nathans et al.,1986B). All of the family of G protein-coupled receptors appear to besimilar to other members of the family of G protein-coupled receptors(e.g., dopaminergic, muscaric, serotonergic, tachykinin, etc.), and eachappears to share the characteristic seven-transmembrane segmenttopography.

When comparing the seven-transmembrane segment receptors with oneanother, a discernible pattern of amino acid sequence conservation isobserved. Transmembrane domains are often the most similar, whereas theamino and carboxyl terminal regions and the cytoplasmic loop connectingtransmembrane segments V and VI can be quite divergent (Dohlman et al.,1987).

Interaction with cytoplasmic polypeptides, such as kinases and Gproteins, was predicted to involve the hydrophobic loops connecting thetransmembrane domains of the receptor. The challenge, however, has beento determine which features are preserved among the seven-transmembranesegment receptors because of conservation of function, and whichdivergent features represent structural adaptations to new functions. Anumber of strategies have been used to test these ideas, including theuse of recombinant DNA and gene expression techniques for theconstruction of substitution and deletion mutants, as well as of hybridor chimeric receptors (Dohlman et al., 1991).

With the growing number of receptor sub-types, G-protein subunits, andeffectors, characterization of ligand binding and G protein recognitionproperties of these receptors is an important area for investigation. Ithas long been known that multiple receptors can couple to a single Gprotein and, as in the case of epinephrine binding to β₂- andα₂-adrenergic receptors, a single ligand can bind to multiplefunctionally distinct receptor sub-types. Moreover, G proteins withsimilar receptor and effector coupling specificities have also beenidentified. For example, three species of human G_(i) have been cloned(Itoh et al., 1988), and alternate mRNA splicing has been shown toresult in multiple variants of G_(s) (Kozasa et al., 1988). Cloning andover production of the muscarinic and α₂-adrenergic receptors led to thedemonstration that a single receptor sub-type, when expressed at highlevels in the cell, will couple to more than one type of G protein.

Opioid receptors are known to be sensitive to reducing agents, and theoccurrence of a disulfide bridge has been postulated as essential forligand binding (Gioannini et al., 1989). For rhodopsin, muscarinic, andβ-adrenergic receptors, two conserved cysteine residues in each of thetwo first extracellular loops have been shown critical for stabilizingthe functional protein structure and are presumed to do so by forming adisulfide bridge. Structure/function studies of opioid ligands haveshown the importance of a protonated amine group for binding to thereceptor with high affinity. The binding site of the receptor might,therefore, possess a critical negatively charged counterpartCatecholamine receptors display in their sequence a conserved aspartateresidue that has been shown necessary for binding the positively chargedamine group of their ligands.

Given the complexity and apparent degeneracy of function of variousopioid receptors, a question of fundamental importance is how, and underwhat circumstances do specific sub-type and sub-sub-type receptors exerttheir physiological effect in the presence of the appropriatestimulatory ligand. A traditional approach to answering this questionhas been to reconstitute the purified receptor and G protein componentsin vitro. Unfortunately, purification schemes have been successful foronly a very limited number of receptor sub-types and their cognateG-proteins. Alternatively, heterologous expression systems can be ofmore general usefulness in the characterization of cloned receptors andin elucidating receptor G protein coupling specificity (Marullo et al.,1988; Payette et al., 1990; King et al., 1990).

One such system was recently developed in yeast cells, in which thegenes for a mammalian β₂-adrenergic receptor and G_(s) α-subunit werecoexpressed (King et al., 1990). Expression of the β₂-adrenergicreceptor to levels several hundred-fold higher than in any human tissuewas attained, and ligand binding was shown to be of the appropriateaffinity, specificity, and stereoselectivity. Moreover, a β₂-adrenergicreceptor-mediated activation of the pheromone signal transductionpathway was demonstrated by several criteria, including imposition ofgrowth arrest, morphological changes, and induction of apheromone-responsive promoter (FUS1) fused to the Escherichia coli lacZgene (encoding β-galactosidase) (King et al., 1990).

Finally, expression of a single receptor in the absence of other relatedsub-types is often impossible to achieve, even in isolated,non-recombinant mammalian cells. Thus, there has been considerabledifficulty in applying the standard approaches of classical genetics oreven the powerful techniques of molecular biology to the study of opioidreceptors. In particular, means are needed for the identification of theDNA sequences encoding individual opioid receptors. Given such isolated,recombinant sequences, it is possible to address the heretoforeintractable problems associated with design and testing ofisoform-specific opioid receptor agonists and antagonists. Theavailability of cDNAs encoding the opioid receptors will permit detailedstudies of signal-transduction mechanisms and reveal the anatomicaldistribution of the mRNAs of these receptors, providing information ontheir expression pattern in the nervous system. This information shouldultimately allow better understanding of the opioid system in analgesia,and also the design of more specific therapeutic drugs.

Availability of polynucleotide sequences encoding opioid receptors, andthe polypeptide sequences of the encoded receptors, will significantlyincrease the capability to design pharmaceutical compositions, such asanalgesics, with enhanced specificity of function. In general, theavailability of these polypeptide sequences will enable efficientscreening of candidate compositions. The principle in operation throughthe screening process is straightforward: natural agonists andantagonists bind to cell-surface receptors and channels to producephysiological effects; certain other molecules bind to receptors andchannels; therefore, certain other molecules may produce physiologicaleffects and act as therapeutic pharmaceutical agents. Thus, the abilityof candidate drugs to bind to opioid receptors can function as anextremely effective screening criterion for the selection ofpharmaceutical compositions with a desired functional efficacy.

Prior methods for screening candidate drug compositions based on theirability to preferentially bind to cell-surface receptors has beenlimited to tissue-based techniques. In these techniques, animal tissuesrich in the receptor type of interest are extracted and prepared;candidate drugs are then allowed to interact with the prepared tissueand those found to bind to the receptors are selected for further study.However, these tissue-based screening techniques suffer from severalsignificant disadvantages. First, they are expensive because the sourceof receptor cell tissue—laboratory animals—is expensive. Second,extensive technical input is required to operate the screens. And,third, the screens may confuse the results because there are no tissueswhere only one receptor subtype is expressed exclusively. Withtraditional prior art screens you are basically looking at the wronginteractions or, at best, the proper interactions mixed in with a wholevariety of unwanted interactions. An additional fundamental deficiencyof animal tissue screens is that they contain animal receptors—ideal forthe development of drugs for animals but of dubious value in humantherapeutic agents.

The solution to this problem provided by the present invention isobvious. A polynucleotide of the present invention, transfected intosuitable host cells, can express polypeptide sequences corresponding toopioid receptors, both in large quantities and through relatively simplelaboratory procedures. The result is the availability of extremelyspecific receptor-drug interactions free from the competitive andunwanted interactions encountered in tissue-based screens. Furtherexpression in a microorganism where no such endogenous receptors exist(e.g. yeast cells or mutant mammalian cell lines) can be useful forscreening and evaluating sub-type-selective drugs (Marullo et a, 1988;Payette et al., 1990; and King et al., 1990).

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides an isolated and purifiedpolynucleotide that encodes a mu opioid receptor polypeptide and atranscription regulatory polypeptide. In a preferred embodiment, apolynucleotide of the present invention is a DNA molecule. Even morepreferred, a polynucleotide of the present invention encodes apolypeptide comprising the amino acid residue sequence of SEQ ID NO:2 orSEQ ID NO:4. Most preferably, an isolated and purified polynucleotide ofthe invention comprises the nucleotide base sequence of SEQ ID NO:1 orSEQ ID NO:3.

Yet another aspect of the present invention contemplates an isolated andpurified polynucleotide comprising a base sequence that is identical orcomplementary to a segment of at least 10 contiguous bases of SEQ IDNO:1 or SEQ ID NO:3, wherein the polynucleotide hybridizes to apolynucleotide that encodes a mu opioid receptor polypeptide.Preferably, an isolated and purified polynucleotide comprises a basesequence that is identical or complementary to a segment of at least 25to 70 contiguous bases of SEQ ID NO:1 or SEQ ID NO:3. For example, apolynucleotide of the invention can comprise a segment of basesidentical or complementary to 40 to 55 contiguous bases of the disclosednucleotide sequences.

In still another embodiment of the present invention, there is providedan isolated and purified polynucleotide comprising a base sequence thatis identical or complementary to a segment of at least 10 contiguousbases of SEQ ID NO:1. The polynucleotide of the invention hybridizes toSEQ ID NO:1, or a complement of SEQ ID NO:1. Preferably, the isolatedand purified polynucleotide comprises a base sequence that is identicalor complementary to a segment of at least 25 to 70 contiguous bases ofSEQ ID NO:1. For example, the polynucleotide of the invention cancomprise a segment of bases identical or complementary to 40 to 55contiguous bases of SEQ ID NO:1.

Alternatively, the present invention contemplates an isolated andpurified polynucleotide that comprises a base sequence that is identicalor complementary to a segment of at least 10 contiguous bases of SEQ IDNO:3. The polynucleotide of the invention hybridizes to SEQ ID NO:3, ora complement of SEQ ID NO:3. Preferably, the polynucleotide comprises abase sequence that is identical or complementary to a segment of atleast 25 to 70 contiguous bases of SEQ ID NO:3. For example, thepolynucleotide can comprise a segment of bases identical orcomplementary to 40 to 55 contiguous bases of SEQ ID NO:3.

In another embodiment, the present invention contemplates an isolatedand purified mu opioid receptor polypeptide or a gene transcriptionregulatory polypeptide. Preferably, a polypeptide of the invention is arecombinant polypeptide. Even more preferably, an opioid receptorpolypeptide of the present invention comprises the amino acid residuesequence of SEQ ID NO:2 and a gene transcription regulatory polypeptideof the present invention comprises the amino acid residue sequence ofSEQ ID NO:4.

In an alternative embodiment, the present invention provides anexpression vector comprising a polynucleotide that encodes a mu opioidreceptor polypeptide. Preferably, an expression vector of the presentinvention comprises a polynucleotide that encodes a polypeptidecomprising the amino acid residue sequence of SEQ ID NO:2 or SEQ IDNO:4. More preferably, an expression vector of the present inventioncomprises a polynucleotide comprising the nucleotide base sequence ofSEQ ID NO:1 or SEQ ID NO:3. Even more preferably, an expression vectorof the invention comprises a polynucleotide operatively linked to anenhancer-promoter. More preferably still, an expression vector of theinvention comprises a polynucleotide operatively linked to a prokaryoticpromoter. Alternatively, an expression vector of the present inventioncomprises a polynucleotide operatively linked to an enhancer-promoterthat is a eukaryotic promoter, and the expression vector furthercomprises a polyadenylation signal that is positioned 3′ of thecarboxyl-terminal amino acid and within a transcriptional unit of theencoded polypeptide.

In yet another embodiment, the present invention provides a recombinanthost cell transfected with a polynucleotide that encodes a mu opioidreceptor polypeptide. Preferably, a recombinant host cell of the presentinvention is transfected with the polynucleotide of SEQ ID NO:1 or SEQID NO:3. Even more preferably, a host cell of the invention is aeukaryotic host cell. Still more preferably, a recombinant host cell ofthe present invention is a yeast cell. Alternatively, a recombinant hostcell of the invention is a COS or CHO cell. In another aspect, arecombinant host cell of the present invention is a prokaryotic hostcell. Preferably, a recombinant host cell of the invention is abacterial cell of the DH5α strain of Escherichia coli. More preferably,a recombinant host cell comprises a polynucleotide under thetranscriptional control of regulatory signals functional in therecombinant host cell, wherein the regulatory signals appropriatelycontrol expression of a mu opioid receptor polypeptide in a manner toenable all necessary transcriptional and post-transcriptionalmodification.

In yet another embodiment, the present invention contemplates a processof preparing a mu opioid receptor polypeptide comprising transfecting acell with polynucleotide that encodes a mu opioid receptor polypeptideto produce a transformed host cell and maintaining the transformed hostcell under biological conditions sufficient for expression of thepolypeptide. Preferably, the transformed host cell is a eukazyotic cell.More preferably still, the eukaryotic cell is a COS or CHO cell.Alternatively, the host cell is a prokaryotic cell. More preferably, theprokaryotic cell is a bacterial cell of the DH5α strain of Escherichiacoli. Even more preferably, a polynucleotide transfected into thetransformed cell comprises the nucleotide base sequence of SEQ ID NO:1or SEQ ID NO:3.

In still another embodiment, the present invention provides an antibodyimmunoreactive with a mu opioid receptor polypeptide. Preferably, anantibody of the invention is a monoclonal antibody. More preferably, amu opioid receptor polypeptide comprises the amino acid residue sequenceof SEQ ID NO:2 or SEQ ID NO:4.

In another aspect, the present invention contemplates a process ofproducing an antibody immunoreactive with a mu opioid receptorpolypeptide comprising the steps of (a) transfecting a recombinant hostcell with a polynucleotide that encodes a mu opioid receptorpolypeptide; (b) culturing the host cell under conditions sufficient forexpression of the polypeptide; (c) recovering the polypeptide; and (d)preparing the antibody to the polypeptide. Preferably, the host cell istransfected with the polynucleotide of SEQ ID NO:1 or SEQ ID NO:3.Alternatively, steps (a), (b) and (c) can be avoided by use of asynthetic polypeptide. Even more preferably, the present inventionprovides an antibody prepared according to the process described above.

Alternatively, the present invention provides a process of detecting amu opioid receptor polypeptide, wherein the process comprisesimmunoreacting the polypeptide with an antibody prepared according tothe process described above to form an antibody-polypeptide conjugate,and detecting the conjugate.

In yet another embodiment, the present invention contemplates a processof detecting a messenger RNA transcript that encodes a mu opioidreceptor polypeptide, wherein the process comprises (a) hybridizing themessenger RNA transcript with a polynucleotide sequence that encodes themu opioid receptor polypeptide to form a duplex; and (b) detecting theduplex. Alternatively, the present invention provides a process ofdetecting a DNA molecule that encodes a mu opioid receptor polypeptide,wherein the process comprises (a) hybridizing DNA molecules with apolynucleotide that encodes a mu opioid receptor polypeptide to form aduplex; and (b) detecting the duplex.

In another aspect, the present invention contemplates a diagnostic assaykit for detecting the presence of a mu opioid receptor polypeptide in abiological sample, where the kit comprises a first container containinga first antibody capable of immunoreacting with a mu opioid receptorpolypeptide, with the first antibody present in an amount sufficient toperform at least one assay. Preferably, an assay kit of the inventionfurther comprises a second container containing a second antibody thatimmunoreacts with the first antibody. More preferably, the antibodiesused in an assay kit of the present invention are monoclonal antibodies.Even more preferably, the first antibody is affixed to a solid support.More preferably still the first and second antibodies comprise anindicator, and, preferably, the indicator is a radioactive label or anenzyme.

In an alternative aspect, the present invention provides a diagnosticassay kit for detecting the presence, in biological samples, of apolynucleotide that encodes a mu opioid receptor polypeptide, the kitcomprising a first container that contains a second polynucleotideidentical or complementary to a segment of at least 10 contiguousnucleotide bases of SEQ ID NO:1 or SEQ ID NO:3.

In another embodiment, the present invention contemplates a diagnosticassay kit for detecting the presence, in a biological sample, of anantibody immunoreactive with a mu opioid receptor polypeptide, the kitcomprising a first container containing a mu opioid receptor polypeptidethat immunoreacts with the antibody, with the polypeptide present in anamount sufficient to perform at least one assay.

In yet another aspect, the present invention contemplates a process ofscreening substances for their ability to interact with a mu opioidreceptor polypeptide comprising the steps of providing a mu opioidreceptor polypeptide, and testing the ability of selected substances tointeract with the opioid receptor polypeptide.

In a preferred embodiment, providing a mu opioid receptor polypeptide istransfecting a host cell with a polynucleotide that encodes a mu opioidreceptor polypeptide to form a transformed cell and maintaining thetransformed cell under biological conditions sufficient for expressionof the opioid receptor polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings which form a portion of the specification it is shownthat in FIG. 1A and FIG. 1B amino acid sequence alignment of MOR-1, themu opioid receptor, with the mouse δ-opioid receptor (DOR-1) (Evans etal., 1992) and the rat somatostatin receptor (SOM1 and SOM2) (Meyerhofet al., 1991; Kluxen et al., 1992). Seven hydrophobic domains areunderlined and numbered I to VII.-, Amino acids identical to those inMOR-1. Spaces, gaps introduced for alignment, diamond figure, putativeN-linked glycosylation sites; downward arrow, potential site forphosphorylation by CAMP-dependent protein kinase; O, potential sites forphosphorylation by protein kinase C; , conserved aspartic acid residuesproposed to interact with the amine group of ligands; =, conservedcysteine residues that might form a disulfide bond; ♦, potentialpahnitoylation site. The sequence for the MOR-1 cDNA has been submittedto GenBank (accession number L13069).

It is shown in FIG. 2 that saturation binding of [³H]diprenorphine usingCOS-7 cell membranes. [³H]Diprenorphine binding was determined usingmembranes prepared from COS-7 cells transfected with either the ratMOR-1 cDNA plasmid () or the parental vector (◯). Data from arepresentative experiment are presented and are expressed as mean ±standard error. Inset, Scatchard plot analysis of the binding data fromMOR-1-transfected cells.

It is shown in FIG. 3A and FIG. 3B that displacement of[³H]diprenorphine binding with unlabeled ligands as competitors. Datafrom a representative experiment are presented for each ligand. Top,using opioid agonists as competitors; bottom, using opioid antagonistsand somatostatins as competitors.

It is shown in FIG. 4 that functional coupling of MOR-1 to adenylylcyclase. Parental COS-7 cells (Nontransfected cells) or COS-7 cellsexpressing MOR-1 (Transfected cells) were stimulated with forskolin(Forsk.) to elevate adenylyl cyclase activity above basal levels. Theμ-selective ligands were included during forskolin treatment asindicated. Cellular cAMP levels were determined. Data are expressed asmean ± standard error (four experiments). *, Data are significantlydifferent from the control group (transfected cells treated withforskolin only).

DETAILED DESCRIPTION OF THE INVENTION I. The Invention

The present invention provides DNA segments, purified polypeptides,methods for obtaining antibodies, methods of cloning and usingrecombinant host cells necessary to obtain and use recombinant mu opioidreceptors. Thus, the difficulties encountered with applying the standardapproaches of classical genetics or techniques in molecular biologyevident in the prior art to mu opioid receptors, have been overcome.Accordingly, the present invention concerns generally compositions andmethods for the preparation and use of mu opioid receptors.

II. Polynucleotide

A. Isolated and Purified Polynucleotides That Encode mu Opioid ReceptorPolypeptides

In one aspect, the present invention provides an isolated and purifiedpolynucleotide that encodes a mu opioid receptor polypeptide. In apreferred embodiment, the polynucleotide of the present invention is aDNA molecule. Even more preferred, a polynucleotide of the presentinvention encodes a polypeptide comprising the amino acid residuesequence of SEQ ID NO:2 or SEQ ID NO:4. Most preferably, an isolated andpurified polynucleotide of the invention comprises the nucleotide basesequence of SEQ ID NO:1 or SEQ ID NO:3.

As used herein, the term “polynucleotide” means a sequence ofnucleotides connected by phosphodiester linkages. Polynucleotides arepresented herein in the direction from the 5′ to the 3′ direction. Apolynucleotide of the present invention can comprise from about 680 toabout several hundred thousand base pairs. Preferably, a polynucleotidecomprises from about 680 to about 150,000 base pairs. Preferred lengthsof particular polynucleotide are set forth hereinafter.

A polynucleotide of the present invention can be a deoxyribonucleic acid(DNA) molecule or ribonucleic acid (RNA) molecule. Where apolynucleotide is a DNA molecule, that molecule can be a gene or a cDNAmolecule. Nucleotide bases are indicated herein by a single letter code:adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) anduracil (U).

A polynucleotide of the present invention can be prepared using standardtechniques well known to one of skill in the art. The preparation of acDNA molecule encoding a mu opioid receptor polypeptide of the presentinvention is described hereinafter in Examples 1 and 2. A polynucleotidecan also be prepared from genomic DNA libraries using lambda phagetechnologies.

In another aspect, the present invention provides an isolated andpurified polynucleotide that encodes a mu opioid receptor polypeptide,where the polynucleotide is preparable by a process comprising the stepsof constructing a library of cDNA clones from a cell that expresses thepolypeptide; screening the library with a labelled cDNA probe preparedfrom RNA that encodes the polypeptide; and selecting a clone thathybridizes to the probe. Preferably, the polynucleotide of the inventionis prepared by the above process. More preferably, the polynucleotide ofthe invention encodes a polypeptide that has the amino acid residuesequence of SEQ ID NO:2 or SEQ ID NO:4. More preferably still, thepolynucleotide comprises the nucleotide sequence of SEQ ID NO:1 or SEQID NO:3.

B. Probes and Primers

In another aspect, DNA sequence information provided by the presentinvention allows for the preparation of relatively short DNA (or RNA)sequences having the ability to specifically hybridize to gene sequencesof the selected polynucleotide disclosed herein. In these aspects,nucleic acid probes of an appropriate length are prepared based on aconsideration of a selected nucleotide sequence, e.g., a sequence suchas that shown in SEQ ID NO:1. The ability of such nucleic acid probes tospecifically hybridize to a polynucleotide encoding a mu opioid receptorlends them particular utility in a variety of embodiments. Mostimportantly, the probes can be used in a variety of assays for detectingthe presence of complementary sequences in a given sample.

In certain embodiments, it is advantageous to use oligonucleotideprimers. The sequence of such primers is designed using a polynucleotideof the present invention for use in detecting, amplifying or mutating adefined segment of a gene or polynucleotide that encodes a mu opioidreceptor polypeptide from mammalian cells using PCR™ technology.

To provide certain of the advantages in accordance with the presentinvention, a preferred nucleic acid sequence employed for hybridizationstudies or assays includes probe molecules that are complementary to atleast a 10 to 70 or so long nucleotide stretch of a polynucleotide thatencodes a mu opioid receptor polypeptide, such as that shown in SEQ IDNOS:1 or 3. A size of at least 10 nucleotides in length helps to ensurethat the fragment will be of sufficient length to form a duplex moleculethat is both stable and selective. Molecules having complementarysequences over stretches greater than 10 bases in length are generallypreferred, though, in order to increase stability and selectivity of thehybrid, and thereby improve the quality and degree of specific hybridmolecules obtained. One will generally prefer to design nucleic acidmolecules having gene-complementary stretches of 25 to 40 nucleotides,55 to 70 nucleotides, or even longer where desired. Such fragments canbe readily prepared by, for example, directly synthesizing the fragmentby chemical means, by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. No. 4,683,202,herein incorporated by reference, or by excising selected DNA fragmentsfrom recombinant plasmids containing appropriate inserts and suitablerestriction enzyme sites.

In another aspect, the present invention contemplates an isolated andpurified polynucleotide comprising a base sequence that is identical orcomplementary to a segment of at least 10 contiguous bases of SEQ IDNO:1 or SEQ ID NO:3, wherein the polynucleotide hybridizes to apolynucleotide that encodes a mu opioid receptor polypeptide.Preferably, the isolated and purified polynucleotide comprises a basesequence that is identical or complementary to a segment of at least 25to 70 contiguous bases of SEQ ID NO:1 or SEQ ID NO:3. For example, thepolynucleotide of the invention can comprise a segment of basesidentical or complementary to 40 or 55 contiguous bases of the disclosednucleotide sequences.

Accordingly, a polynucleotide probe molecule of the invention can beused for its ability to selectively form duplex molecules withcomplementary stretches of the gene. Depending on the applicationenvisioned, one will desire to employ varying conditions ofhybridization to achieve varying degree of selectivity of the probetoward the target sequence. For applications requiring a high degree ofselectivity, one will typically desire to employ relatively stringentconditions to form the hybrids. For example, one will select relativelylow salt and/or high temperature conditions, such as provided by0.02M-0.15M NaCl at temperatures of 50° C. to 70° C. Those conditionsare particularly selective, and tolerate little, if any, mismatchbetween the probe and the template or target strand.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate a mu opioid receptorpolypeptide coding sequence from other cells, functional equivalents, orthe like, less stringent hybridization conditions are typically neededto allow formation of the heteroduplex. In these circumstances, one candesire to employ conditions such as 0.15M-0.9M salt, at temperaturesranging from 20° C. to 70° C. Cross-hybridizing species can thereby bereadily identified as positively hybridizing signals with respect tocontrol hybridizations. In any case, it is generally appreciated thatconditions can be rendered more stringent by the addition of increasingamounts of formamide, which serves to destabilize the hybrid duplex inthe same manner as increased temperature. Thus, hybridization conditionscan be readily manipulated, and thus will generally be a method ofchoice depending on the desired results.

In still another embodiment of the present invention, there is providedan isolated and purified polynucleotide comprising a base sequence thatis identical or complementary to a segment of at least 10 contiguousbases of SEQ ID NO:1. The polynucleotide of the invention hybridizes toSEQ ID NO:1, or a complement of SEQ ID NO:1. Preferably, the isolatedand purified polynucleotide comprises a base sequence that is identicalor complementary to a segment of at least 25 to 70 contiguous bases ofSEQ ID NO:1. For example, the polynucleotide of the invention cancomprise a segment of bases identical or complementary to 40 to 55contiguous bases of SEQ ID NO:1.

Alternatively, the present invention contemplates an isolated andpurified polynucleotide that comprises a base sequence that is identicalor complementary to a segment of at least 10 contiguous bases of SEQ IDNO:3. The polynucleotide of the invention hybridizes to SEQ ID NO:3, ora complement of SEQ ID NO:3. Preferably, the polynucleotide comprises abase sequence that is identical or complementary to a segment of atleast 25 to 70 contiguous bases of SEQ ID NO:3. For example, thepolynucleotide can comprise a segment of bases identical orcomplementary to 40 to 55 contiguous bases of SEQ ID NO:3.

In certain embodiments, it is advantageous to employ a polynucleotide ofthe present invention in combination with an appropriate label fordetecting hybrid formation A wide variety of appropriate labels areknown in the art, including radioactive, enzymatic or other ligands,such as avidin/biotin, which are capable of giving a detectable signal.

In general, it is envisioned that a hybridization probe described hereinis useful both as a reagent in solution hybridization as well as inembodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed nucleic acid is then subjected tospecific hybridization with selected probes under desired conditions.The selected conditions depend as is well known in the art on theparticular circumstances and criteria required (e.g., on the G+Ccontent, type of target nucleic acid, source of nucleic acid, size ofhybridization probe). Following washing of the matrix to removenon-specifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label.

II. Mu Opioid Receptor Polypeptide and Gene Transcription RegulatoryPolypeptide

In one embodiment, the present invention contemplates an isolated andpurified mu opioid receptor polypeptide. Preferably, a mu opioidreceptor polypeptide of the invention is a recombinant polypeptide. Evenmore preferably, a mu opioid receptor polypeptides of the presentinvention comprises the amino acid residue sequence of SEQ ID NO:2. A muopioid receptor polypeptide preferably comprises less than about 500amino acid residues and, more preferably less than about 400 amino acidresidues.

In another embodiment, the present invention contemplates an isolatedand purified gene transcription regulatory polypeptide. Preferably, agene transcription regulatory polypeptide of the invention is arecombinant polypeptide. Even more preferably, gene transcriptionregulatory polypeptides of the present invention comprises the aminoacid residue sequence of SEQ ID NO:4. A gene transcription regulatorypolypeptide preferably comprises less than about 500 amino acid residuesand, more preferably less than about 400 amino acid residues.

Polypeptides are disclosed herein as amino acid residue sequences. Thosesequences are written left to right in the direction from the amino tothe carboxyl terminus. In accordance with standard nomenclature, aminoacid residue sequences are denominated by either a single letter or athree letter code as indicated below.

Amino Acid Residue 3-Letter Code 1-Letter Code Alanine Ala A ArginineArg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamine GlnQ Glutamic Acid Glu E Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr YValine Val V

Modifications and changes can be made in the structure of a polypeptideof the present invention and still obtain a molecule having like opioidreceptor characteristics. For example, certain amino acids can besubstituted for other amino acids in a sequence without appreciable lossof receptor activity. Because it is the interactive capacity and natureof a polypeptide that defines that polypeptide's biological functionalactivity, certain amino acid sequence substitutions can be made in apolypeptide sequence (or, of course, its underlying DNA coding sequence)and nevertheless obtain a polypeptide with like properties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art (Kyte, J. and R. F. Doolittle 1982). It is knownthat certain amino acids can be substituted for other amino acids havinga similar hydropathic index or score and still result in a polypeptidewith similar biological activity. Each amino acid has been assigned ahydropathic index on the basis of its hydrophobicity and chargecharacteristics. Those indices are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines the interaction of the polypeptide with other molecules,such as enzymes, substrates, receptors, antibodies, antigens, and thelike. It is known in the art that an amino acid can be substituted byanother amino acid having a similar hydropathic index and still obtain afunctionally equivalent polypeptide. In such changes, the substitutionof amino acids whose hydropathic indices are within ±2 is preferred,those which are within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly where the biological functional equivalentpolypeptide or peptide thereby created is intended for use inimmunological embodiments. U.S. Pat. No. 4,554,101, incorporated hereinby reference, states that the greatest local average hydrophilicity of apolypeptide, as governed by the hydrophilicity of its adjacent aminoacids, correlates with its immunogenicity and antigenicity, i.e. with abiological property of the polypeptide.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); proline (−0.5±1);threonine (−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent polypeptide. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those which are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, bydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine (See Table 1, below). The present invention thuscontemplates functional or biological equivalents of a mu opioidreceptor polypeptide as set forth above.

TABLE 1 Original Residue Exemplary Substitutions Ala Gly; Ser Arg LysAsn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala His Asn; Gln IleLeu; Val Leu Ile; Val Lys Arg Met Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Biological or functional equivalents of a polypeptide can also beprepared using site-specific mutagenesis. Site-specific mutagenesis is atechnique useful in the preparation of second generation polypeptides,or biologically functional equivalent polypeptides or peptides, derivedfrom the sequences thereof, through specific mutagenesis of theunderlying DNA. As noted above, such changes can be desirable whereamino acid substitutions are desirable. The technique further provides aready ability to prepare and test sequence variants, for example,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by Adelman et al., (1983). As will beappreciated, the technique typically employs a phage vector which canexist in both a single stranded and double stranded form. Typicalvectors useful in site-directed mutagenesis include vectors such as theM13 phage (Messing et al., 1981). These phage are commercially availableand their use is generally known to those of skill in the art.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector which includeswithin its sequence a DNA sequence which encodes all or a portion of themu opioid receptor polypeptide sequence selected. An oligonucleotideprimer bearing the desired mutated sequence is prepared, generallysynthetically, for example, by the method of Crea et al., (1978). Thisprimer is then annealed to the singled-stranded vector, and extended bythe use of enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cellssuch as E. coli cells and clones are selected which include recombinantvectors bearing the mutation. Commercially available kits come with allthe reagents necessary, except the oligonucleotide primers.

Amino acid residues can be added to or deleted from the mu opioidreceptor polypeptide through the use of standard molecular biologicaltechniques without altering the functionality of the receptor. Forexample, portions of the mu opioid receptor can be removed to createtruncated opioid receptors. The truncated receptor retains theproperties of mu opioid receptors such as ligand binding and the abilityto interact with other proteins (G proteins, adenylyl cyclase, forexample). Functional truncated proteins have been reported forphosphodiesterases, ion channels, and membrane transporters. As usedherein, truncated receptors are receptors in which amino acids have beenremoved from the wild type receptor to create a shorter receptor orportions thereof. As used herein, chimeric receptors are receptors inwhich amino acids have been added to the receptor. A chimeric receptorcan be shorter, longer or the same length as the wild type receptor.

The functional activity of truncated and chimeric receptors have beendemonstrated in a number of receptor systems. In particular, truncatedand chimeric adrenergic receptors, which are structurally similar to theopioid receptors, have been shown to retain functional properties of thewild type adrenergic receptor.

Most of the long carboxyl terminus of the avian β-adrenergic receptorcan be deleted or proteolytically removed without altering theligand-binding properties or regulatory properties of the receptor. Theligand binding properties of five truncated β-adrenergic receptors forboth agonists and antagonists were found to be similar to those of thewild type receptor. Furthermore, truncated adrenergic receptors alsostimulated adenylyl cyclase activity. In fact, truncated β-adrenergicreceptors, in the presence of agonists, showed a greater stimulation ofadenylyl cyclase activity than the stimulation achieved by the wild typereceptor. (Parker et al., 1991).

Similar results were obtained for the α-adrenergic receptor. A truncatedα-adrenergic receptor activated phosphatidyl inositol hydrolysis aseffectively as wild type a-adrenergic receptor. (Cotecchia et al.,1989).

Functional chimeric receptors have also been created by a number ofinvestigators. Functional chimeric adrenergic receptors were created bysplicing together sections of the α₂ and β₂ adrenergic receptors.(Kobilka et al., 1988). Functional chimeras have also been generated forthe following receptors: between β₁ and β₂ receptors, (Fnelle et al.,1988; Marullo et al., 1990); between m2 and m3 muscarinic receptors,(Wess et al. 1990); between m1 muscarinic and β adrenergic receptors,(Wong et al., (1990); between D₂ dopamine and m1 muscarinic receptors,(England et al., 1991); between luteinizing hormone and β adrenergicreceptors, (Moyle et al., 1991); between NK₁ and NK₃ substance Preceptors, (Gether et al., 1993); and platelet-derived growth factor andepidermal growth factor receptors, (Seedorf et al., 1991).

Chimeric mu opioid receptors can be created by splicing sections of asecond receptor to a mu receptor. The two receptors can be similar toeach other. Thus, for the creation of chimeric mu opioid receptors,other opioid receptors, such as sigma, delta, and kappa opioidreceptors, are ideal sources for nucleotide sequences. For example, atransmembrane domain in the mu opioid receptor can be substituted withan analogous transmembrane domain from sigma, delta or kappa opioidreceptor. It is contemplated that the nucleotide source of the secondreceptor is not limited to opioid receptors. Chimeric receptors can becreated from mu opioid receptor and other similar receptors such asacetylcholine, adenosine, adrenergic, angiotensin, bombesin, bradykinin,cannabinoid, dopamine, endothelin, histamine, interleukin, luteinizinghormone, neuromedin K, neuropeptide Y, odorant, prostaglandin,parathyroid hormone, serotonin, somatostatin, substance K, substance P,thrombin, thromboxane A2, thyrotropin releasing hormone and vasopressinreceptors.

A mu opioid receptor polypeptide of the present invention is understoodnot to be limited to a particular source. As disclosed herein, thetechniques and compositions of the present invention provide, forexample, the identification and isolation of mu opioid receptors fromrodent sources. Thus, the invention provides for the general detectionand isolation of the genus of mu opioid receptor polypeptides from avariety of sources. It is believed that a number of species of thefamily of mu opioid receptor polypeptides are amenable to detection andisolation using the compositions and methods of the present inventions.

A polypeptide of the present invention is prepared by standardtechniques well known to those skilled in the art. Such techniquesinclude, but are not limited to, isolation and purification from tissuesknown to contain that polypeptide, and expression from cloned DNA thatencodes such a polypeptide using transformed cells (See Examples 1 and2, hereinafter).

Opioid receptor polypeptides are found in virtually all mammalsincluding human. The sequence of a mouse delta opioid receptor has beenpreviously described (Kieffer et al., 1992; Evans et al., 1992). As isthe case with other receptors, there is likely little variation betweenthe structure and function of an opioid receptor in different species.Where there is a difference between species, identification of thosedifferences is well within the skill of an artisan. Thus, the presentinvention contemplates a mu opioid receptor polypeptide from any mammal.A preferred mammal is a rodent or a human.

Regulation of gene expression in a cell is accomplished through manydifferent mechanisms. A well known mechanism of gene expressionregulation is through the use of a zinc finger motif in a transcriptionregulatory polypeptide. The zinc finger domain found in manytranscription factors binds to DNA to regulate transcription. Zincfinger domains are nucleic acid-binding protein structures firstidentified in the Xenopus transcription factor TFIIIA. These domainshave since been found in numerous nucleic acid-binding proteins. (KlugA. and D. Rhodes, 1987; Evans, R. M. and S. M. Hollenberg, 1988; Payre,F., and A. Vincent, 1988; Miller, J. et al., 1985; Berg J. M., 1988).

A zinc finger domain is composed of 25 to 30 amino acid residues. Thereare two cysteine or histidine residues at both extremities of thedomain, which are most probably involved in the tetrahedral coordinationof a zinc atom Each zinc finger likely binds to the major groove ofB-DNA so as to interact with ˜5 successive base pairs; that is, withabout a half-turn of B-DNA. A zinc finger protein thus can bind to DNAin which the protein binds along one face of the DNA with successivezinc fingers bound in the major groove on alternate sides of the doublehelix. Zinc fingers likely form structural “scaffolds” that match thedouble helix's three dimensional contour. Base sequence specificity ispresumably provided by the particular sequence of each zinc finger'svariable residues. (Klug, A and D. Rhodes, 1987). A schematicrepresentation of a zinc finger domain is shown on the next page:

Zinc fingers have been identified in many transcription factorsincluding Sp1, estrogen, and glucocorticoid receptors, severalDrosophila developmental regulators, and the Xenopus Xfin protein, aswell as in the E. coli UvrA protein and certain retroviral nucleic acidbinding proteins.

Xenopus transcription factor IIIA (TFIIIA) is a regulatory protein whichcontains nine zinc fingers. The 344-residue TFIIIA contains 9 similar,tandemly repeated, ˜30-residue units, each of which contains twoinvariant cysteine residues, two invariant histidine residues, andseveral conserved hydrophobic residues. Each of these units binds a Zn²⁺ion. X-ray absorption measurements indicate that the Zn²⁺ ion istetrahedrally coordinated to the invariant cysteine and histidineresidues. Sequence analysis of a number of transcription regulators hasrevealed that the zinc finger motif occurs between about 2 to 40 timesin a transcription regulator.

Two major classes of zinc fingers are characterized according to thenumber and positions of the histidine and cysteine residues involved inthe zinc atom coordination. In the first class, called C2H2, the firstpair of zinc coordinating residues are cysteines, while the second pairare histidines. Transcription factor TFIIIA is the prototype example forthis class of zinc fingers. A number of experimental reports havedemonstrated the zinc-dependent DNA or RNA binding property of somemembers of this class. The other class of zinc fingers, called C4,groups together many different regulatory proteins that happen to haveseveral cysteines within a short stretch of sequence. The steroidhormone receptors are an example of proteins belonging to this class.

Some of the proteins which are known to include C2H2-type zinc fingersare listed below. We have indicated, between brackets, the number ofzinc finger regions found in each of these proteins; a ‘+’ symbolindicates that only partial sequence data is available and thatadditional finger domains may be present.

Xenopus: transcription factor TFIIIA (9), Xfin (37), XlcOF10 (7),XlcOF22 (12).

Drosophila: Glass (5), Hunchback (6), Kruppel (5), Kruppel-H (4+),Snail(5), Serependity locus beta (6), delta (7), and h-1 (8), Suppressorof hairy wing su(Hw) (12), Tramtrack (2).

Yeast: transcriptional activator ADR1 (2), transcriptional factor SWI5(3).

Aspergillus nidulans: developmental protein br1A (2).

Mammalian: transcription factor Sp1 (3), ZfX (13), ZfY (13), Zfp-35(18), EGR1/Krox24 (3), EGR2/Krox20 (3), Evi-1 (10), GLI1 (5), GLI2(4+),GLI3 (3+), KR1 (9+), KR2 (9), KR3 (15+), KR4 (14+), KR5 (11+),HF.10 (10), HF.12 (6+).

Sequence analysis of rat mu opioid receptor reveals that in an alternatereading frame, the cDNA of the mu opioid receptor (SEQ. ID NO:1) codesfor a polypeptide which contains a zinc finger motif (SEQ. ID NO:3 andSEQ. ID NO:4). The zinc finger containing polypeptide comprises 298amino acids encoded by nucleotides 339 to 1235. The zinc fingercontaining polypeptide is smaller by 100 amino acids than the mu opioidreceptor. SEQ. ID NO:3 shows the alternate reading frame of a mu opioidreceptor that encodes the transcription regulatory polypeptide. Inparticular, there is a zinc finger motif, of the C2H2 cass, locatedbetween amino acid residues 155 and 178 of this protein. This motif fitsthe consensus pattern of C-x(2,4)-C-x(12)-H-x(3,5)-H for the C2H2 class,with 4 amino acid residues each in between the two cysteines at theamino end of the motif and the two histidines at the carboxyl end of themotif. The C2H2 zinc finger motif has been found in many proteins,including mammalian transcription factor Sp1 as discussed above.

It is likely that the zinc finger polypeptide of the mu opioid receptoris involved in the autoregulation of the expression of the mu opioidreceptor. The polynucleotide that encodes the zinc finger polypeptideand the gene transcription regulatory polypeptide is useful incontrolling the expression of the mu opioid receptor. An antibodyimmunoreactive with the gene transcription regulatory polypeptide can beused to regulate the expression of the mu opioid receptor.Alternatively, anti-sense mRNA can be used to regulate the expression ofthe mu opioid receptor.

In another embodiment, the polynucleotide that encodes the genetranscription regulatory polypeptide can be used to identify otherpolynucleotides that encode a mu opioid receptor or a transcriptionregulatory polypeptide.

III. Expression Vectors

In an alternate embodiment, the present invention provides expressionvectors comprising polynucleotide that encode mu opioid receptorpolypeptides, or a polynucleotide that encodes a gene transcriptionregulatory polypeptide. Preferably, expression vectors of the presentinvention comprise polynucleotides that encode polypeptides comprisingthe amino acid residue sequence of SEQ ID NO:2 or SEQ ID NO:4. Morepreferably, expression vectors of the present invention comprisepolynucleotides comprising the nucleotide base sequence of SEQ ID NO:1or SEQ ID NO:3. Even more preferably, expression vectors of theinvention comprise polynucleotides operatively linked to anenhancer-promoter. More preferably still, expression vectors of theinvention comprise a polynucleotide operatively linked to a prokaryoticpromoter. Alternatively, expression vectors of the present inventioncomprise a polynucleotide operatively linked to an enhancer-promoterthat is a eukaryotic promoter. Expression vectors further comprise apolyadenylation signal that is positioned 3′ of the carboxyl-terminalamino acid and within a transcriptional unit of the encoded polypeptide.

A promoter is a region of a DNA molecule typically within about 100nucleotide pairs in front of (upstream of) the point at whichtranscription begins (i.e., a transcription start site). That regiontypically contains several types of DNA sequence elements that arelocated in similar relative positions in different genes. As usedherein, the term “promoter” includes what is referred to in the art asan upstream promoter region, a promoter region or a promoter of ageneralized eukaryotic RNA Polymerase II transcription unit.

Another type of discrete transcription regulatory sequence element is anenhancer. An enhancer provides specificity of time, location andexpression level for a particular encoding region (e.g., gene). A majorfunction of an enhancer is to increase the level of transcription of acoding sequence in a cell that contains one or more transcriptionfactors that bind to that enhancer. Unlike a promoter, an enhancer canfunction when located at variable distances from transcription startsites so long as a promoter is present.

As used herein, the phrase “enhancer-promote” means a composite unitthat contains both enhancer and promoter elements. An enhancer-promoteris operatively linked to a coding sequence that encodes at least onegene product. As used herein, the phrase “operatively linked” means thatan enhancer-promoter is connected to a coding sequence in such a waythat the transcription of that coding sequence is controlled andregulated by that enhancer-promoter. Means for operatively linking anenhancer-promoter to a coding sequence are well known in the art. As isalso well known in the art, the precise orientation and locationrelative to a coding sequence whose transcription is controlled, isdependent inter alia upon the specific nature of the enhancer-promoter.Thus, a TATA box minimal promoter is typically located from about 25 toabout 30 base pairs upstream of a transcription initiation site and anupstream promoter element is typically located from about 100 to about200 base pairs upstream of a transcription initiation site. In contrast,an enhancer can be located downstream from the initiation site and canbe at a considerable distance from that site.

An enhancer-promoter used in a vector construct of the present inventioncan be any enhancer-promoter that drives expression in a cell to betransfected. By employing an enhancer-promoter with well-knownproperties, the level and pattern of gene product expression can beoptimized.

A coding sequence of an expression vector is operatively linked to atranscription terminating region. RNA polymerase transcribes an encodingDNA sequence through a site where polyadenylation occurs. Typically, DNAsequences located a few hundred base pairs downstream of thepolyadenylation site serve to terminate transcription. Those DNAsequences are referred to herein as transcription-termination regions.Those regions are required for efficient polyadenylation of transcribedmessenger RNA (RNA). Transcription-terminating regions are well known inthe art. A preferred transcription-terminating region is derived from abovine growth hormone gene.

An expression vector comprises a polynucleotide that encodes a mu opioidreceptor polypeptide. Such a polypeptide is meant to include a sequenceof nucleotide bases encoding a mu opioid receptor polypeptide sufficientin length to distinguish said segment from a polynucleotide segmentencoding a non-opioid receptor polypeptide. A polypeptide of theinvention can also encode biologically functional polypeptides orpeptides which have variant amino acid sequences, such as with changesselected based on considerations such as the relative hydropathic scoreof the amino acids being exchanged. These variant sequences are thoseisolated from natural sources or induced in the sequences disclosedherein using a mutagenic procedure such as site-directed mutagenesis.

Preferably, expression vectors of the present invention comprisepolynucleotides that encode polypeptides comprising the amino acidresidue sequence of SEQ ID NO:2 or SEQ ID NO:4. An expression vector caninclude a mu opioid receptor polypeptide coding region itself of any ofthe mu opioid receptor polypeptides noted above or it can contain codingregions bearing selected alterations or modifications in the basiccoding region of such a mu opioid receptor polypeptide. Alternatively,such vectors or fragments can code larger polypeptides or polypeptideswhich nevertheless include the basic coding region. In any event, itshould be appreciated that due to codon redundancy as well as biologicalfunctional equivalence, this aspect of the invention is not limited tothe particular DNA molecules corresponding to the polypeptide sequencesnoted above.

Exemplary vectors include the mammalian expression vectors of the pCMVfamily including pCMV6b and pCMV6c (Chiron Corp., Emeryville Calif.) andpRc/CMV (Invitrogen, San Diego, Calif.). In certain cases, andspecifically in the case of these individual mammalian expressionvectors, the resulting constructs can require co-transfection with avector containing a selectable marker such as pSV2neo. Viaco-transfection into a dihydrofolate reductase-deficient Chinese hamsterovary cell line, such as DG44, clones expressing opioid polypeptides byvirtue of DNA incorporated into such expression vectors can be detected.

A DNA molecule of the present invention can be incorporated into avector using a number of techniques which are well known in the art. Forinstance, the vector pUC18 has been demonstrated to be of particularvalue. Likewise, the related vectors M13mp18 and M13mp19 can be used incertain embodiments of the invention, in particular, in performingdideoxy sequencing.

An expression vector of the present invention is useful both as a meansfor preparing quantities of the mu opioid receptor polypeptide-encodingDNA itself, and as a means for preparing the encoded polypeptides. It iscontemplated that where mu opioid receptor polypeptides of the inventionare made by recombinant means, one can employ either prokaxyotic oreukaryotic expression vectors as shuttle systems. However, in thatprokaryotic systems are usually incapable of correctly processingprecursor polypeptides and, in particular, such systems are incapable ofcorrectly processing membrane associated eukaryotic polypeptides, andsince eukazyotic mu opioid receptor polypeptides are anticipated usingthe teaching of the disclosed invention, one likely expresses suchsequences in eukaryotic hosts. However, even where the DNA segmentencodes a eukaryotic mu opioid receptor polypeptide, it is contemplatedthat prokaryotic expression can have some additional applicability.Therefore, the invention can be used in combination with vectors whichcan shuttle between the eukaryotic and prokaryotic cells. Such a systemis described herein which allows the use of bacterial host cells as wellas eukaryotic host cells.

Where expression of recombinant polypeptide of the present invention isdesired and a eukaryotic host is contemplated, it is most desirable toemploy a vector, such as a plasmid, that incorporates a eukaryoticorigin of replication. Additionally, for the purposes of expression ineukaryotic systems, one desires to position the opioid receptor encodingsequence adjacent to and under the control of an effective eukaryoticpromoter such as promoters used in combination with Chinese hamsterovary cells. To bring a coding sequence under control of a promoter,whether it is eukaryotic or prokaryotic, what is generally needed is toposition the 5′ end of the translation initiation side of the propertranslational reading frame of the polypeptide between about 1 and about50 nucleotides 3′ of or downstream with respect to the promoter chosen.Furthermore, where eukaryotic expression is anticipated, one wouldtypically desire to incorporate into the transcriptional unit whichincludes the mu opioid receptor polypeptide, an appropriatepolyadenylation site.

The pRc/CMV vector (available from Invitrogen) is an exemplary vectorfor expressing a mu opioid receptor or a gene transcription regulatorypolypeptide in mammalian cells, particularly COS and CHO cells. Apolypeptide of the present invention under the control of a CMV promotercan be efficiently expressed in mammalian cells. A detailed descriptionof using and expressing a mu opioid receptor in the vector pRc/CMV isprovided in examples 2 and 3 of the present application.

pCMV vectors is another exemplary vector. The pCMV plasmids are a seriesof mammalian expression vectors of particular utility in the presentinvention. The vectors are designed for use in essentially all culturedcells and work extremely well in SV40-transformed simian COS cell lines.The pCMV1, 2, 3, and 5 vectors differ from each other in certain uniquerestriction sites in the polylinker region of each plasmid. The pCMV4vector differs from these 4 plasmids in containing a translationenhancer in the sequence prior to the polylinker. While they are notdirectly derived from the pCMV1-5 series of vectors, the functionallysimilar pCMV6b and c vectors are available from the Chiron Corp. ofEmeryville, Calif. and are identical except for the orientation of thepolylinker region which is reversed in one relative to the other.

The universal components of the pCMV plasmids are as follows. The vectorbackbone is pTZ18R (Pharmacia), and contains a bacteriophage f1 originof replication for production of single stranded DNA and anampicillin-resistance gene. The CMV region consists of nucleotides −760to +3 of the powerful promoter-regulatory region of the humancytomegalovirus (Towne stain) major immediate early gene (Thonsen etal., 1984; Boshart et al., 1985). The human growth hormone fragment(hGH) contains transcription termination and poly-adenylation signalsrepresenting sequences 1533 to 2157 of this gene (Seeburg, 1982). Thereis an Alu middle repetitive DNA sequence in this fragment Finally, theSV40 origin of replication and early region promoter-enhancer derivedfrom the pcD-X plasmid (HindIII to PstI fragment) described in Okayamaet al., (1983). The promoter in this fragment is oriented such thattranscription proceeds away from the CMV/hGH expression cassette.

The pCMV plasmids are distinguishable from each other by differences inthe polylinker region and by the presence or absence of the translationenhancer. The stating pCMV1 plasmid has been progressively modified torender an increasing number of unique restriction sites in thepolylinker region. To create pCMV2, one of two EcoRI sites in pCMV1 weredestroyed. To create pCMV3, pCMV1 was modified by deleting a shortsegment from the SV40 region (StuI to EcoRI), and in so doing madeunique the PstI, SalI, and BamHI sites in the polylinker. To createpCMV4, a synthetic fragment of DNA corresponding to the 5′-untranslatedregion of a mRNA transcribed from the CMV promoter was added C. Thesequence acts as a translational enhancer by decreasing the requirementsfor initiation factors in polypeptide synthesis (Jobling et al., 1987;Browning et al., 1988). To create pCMV5, a segment of DNA (HpaI toEcoRI) was deleted from the SV40 origin region of pCMV1 to render uniqueall sites in the starting polylinker.

The pCMV vectors have been successfully expressed in simian COS cells,mouse L cells, CHO cells, and HeLa cells. In several side by sidecomparisons they have yielded 5- to 10-fold higher expression levels inCOS cells than SV40-based vectors. The pCMV vectors have been used toexpress the LDL receptor, nuclear factor 1, G_(s) alpha polypeptide,polypeptide phosphatase, synaptophysin, synapsin, insulin receptor,influenza hemagglutinin, androgen receptor, sterol 26-hydroxylase,steroid 17- and 21-hydroxylase, cytochrome P-450 oxidoreductase,beta-adrenergic receptor, folate receptor, cholesterol side chaincleavage enzyme, and a host of other cDNAs. It should be noted that theSV40 promoter in these plasmids can be used to express other genes suchas dominant selectable markers. Finally, there is an ATG sequence in thepolylinker between the HindIII and PstI sites in pCMV that can causespurious translation initiation. This codon should be avoided ifpossible in expression plasmids. A paper describing the construction anduse of the parenteral pCMV1 and pCMV4 vectors has been published(Anderson et al., 1989b).

IV. Transfected Cells

In yet another embodiment, the present invention provides recombinanthost cells transformed or transfected with a polynucleotide that encodesa mu opioid receptor polypeptide or transcription regulatorypolypeptide, as well as transgenic cells derived from those transformedor transfected cells. Preferably, recombinant host cells of the presentinvention are transfected with polynucleotide of SEQ ID NO:1 or SEQ IDNO:3. Means of transforming or transfecting cells with exogenouspolynucleotide such as DNA molecules are well known in the art andinclude techniques such as calcium-phosphate- or DEAE-dextran-mediatedtransfection, protoplast fusion, electroporation, liposome mediatedtransfection, direct microinjection and adenovirus infection (Sabrook,Fritsch and Maniatis, 1989).

The most widely used method is transfection mediated by either calciumphosphate or DEAE-dextran. Although the mechanism remains obscure, it isbelieved that the transfected DNA enters the cytoplasm of the cell byendocytosis and is transported to the nucleus. Depending on the celltype, up to 90% of a population of cultured cells can be transfected atany one time. Because of its high efficiency, transfection mediated bycalcium phosphate or DEAE-dextran is the method of choice forexperiments that require transient expression of the foreign DNA inlarge numbers of cells. Calcium phosphate-mediated transfection is alsoused to establish cell lines that integrate copies of the foreign DNA,which are usually arranged in head-to-tail tandem arrays into the hostcell genome.

In the protoplast fusion method, protoplasts derived from bacteriacarrying high numbers of copies of a plasmid of interest are mixeddirectly with cultured mammalian cells. After fusion of the cellmembranes (usually with polyethylene glycol), the contents of thebacteria are delivered into the cytoplasm of the mammalian cells and theplasmid DNA is transported to the nucleus. Protoplast fusion is not asefficient as transfection for many of the cell lines that are commonlyused for transient expression assays, but it is useful for cell lines inwhich endocytosis of DNA occurs inefficiently. Protoplast fusionfrequently yields multiple copies of the plasmid DNA tandemly integratedinto the host chromosome.

The application of brief, high-voltage electric pulses to a variety ofmammalian and plant cells leads to the formation of nanometer-sizedpores in the plasma membrane. DNA is taken directly into the cellcytoplasm either through these pores or as a consequence of theredistribution of membrane components that accompanies closure of thepores. Electroporation can be extremely efficient and can be used bothfor transient expression of cloned genes and for establishment of celllines that carry integrated copies of the gene of interest.Electroporation, in contrast to calcium phosphate-mediated transfectionand protoplast fusion, frequently gives rise to cell lines that carryone, or at most a few, integrated copies of the foreign DNA.

Liposome transfection involves encapsulation of DNA and RNA withinliposomes, followed by fusion of the liposomes with the cell membrane.The mechanism of how DNA is delivered into the cell is unclear buttransfection efficiencies can be as high as 90%.

Direct microinjection of a DNA molecule into nuclei has the advantage ofnot exposing DNA to cellular compartments such as low-pH endosomes.Microinjection is therefore used primarily as a method to establishlines of cells that carry integrated copies of the DNA of interest.

The use of adenovirus as a vector for cell transfection is well known inthe art. Adenovirus vector-mediated cell transfection has been reportedfor various cells (Stratford-Perricaudet et al., 1992).

A transfected cell can be prokaryotic or eukaryotic. Preferably, thehost cells of the invention are eukaryotic host cells. More preferably,the recombinant host cells of the invention are COS cells. Where it isof interest to produce a human mu opioid receptor polypeptides, culturedmammalian or human cells are of particular interest.

In another aspect, the recombinant host cells of the present inventionare prokaryotic host cells. Preferably, the recombinant host cells ofthe invention are bacterial cells of the DH5a strain of Escherichiacoli. In general, prokaryotes are preferred for the initial cloning ofDNA sequences and constructing the vectors useful in the invention. Forexample, E. coli K12 strains can be particularly useful. Other microbialstrains which can be used include E. coli B, and E. coli X1776 (ATCC No.31537). These examples are, of course, intended to be illustrativerather than limiting.

Prokaryotes can also be used for expression. The aforementioned strains,as well as E. coli W3110 (F, λ, prototrophic, ATCC No. 273325), bacillisuch as Bacillus subtilis, or other enterobacteriaceae such asSalmonella typhimurium or Serratia Marcesceus, and various Pseudomonasspecies can be used.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli can betransformed using pBR322, a plasmid derived from an E. coli species(Bolivar et al., 1977). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides easy means for identifyingtransformed cells. The pBR plasmid, or other microbial plasmid or phagemust also contain, or be modified to contain, promoters which can beused by the microbial organism for expression of its own polypeptides.

Those promoters most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase) and lactose promoter systems(Chang et al., 1978; Itakura et al., 1977; Goeddel et al., 1979; Goeddelet al., 1980) and a tryptophan (TRP) promoter system (EPO Appl. Publ.No. 0036776; Siebwenlist et al., 1980). While these are the mostcommonly used, other microbial promoters have been discovered andutilized, and details concerning their nucleotide sequences have beenpublished, enabling a skilled worker to introduce functional promotersinto plasmid vectors (Siebwenlist et al., 1980).

In addition to prokaryotes, eukaryotic microbes, such as yeast can alsobe used. Saccharomyces cerevisiae or common baker's yeast is the mostcommonly used among eukaryotic microorganisms, although a number ofother strains are commonly available. For expression in Saccharomyces,the plasmid YRp7, for example, is commonly used (Stinchcomb et al.,1979; Kingsman et al., 1979; Tschemper et al., 1980). This plasmidalready contains the trpl gene which provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan, forexample ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpllesion as a characteristic of the yeast host cell genome then providesan effective environment for detecting transformation by growth in theabsence of tryptophan.

Suitable promoter sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., 1980) or other glycolyticenzymes (Hess et al., 1968; Holland et al., 1978) such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also introduced into the expression vector downstream from thesequences to be expressed to provide polyadenylation of the mRNA andtermination. Other promoters, which have the additional advantage oftranscription controlled by growth conditions are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utlization Any plasmid vectorcontaining a yeast-compatible promoter, origin or replication andtermination sequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms can also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years (Kruse and Peterson, 1973). Examplesof such useful host cell lines are AtT-20, VERO and HeLa cells, Chinesehamster ovary (CHO) cell lines, and W138, BHK, COSM6, COS-1, COS-7, 293and MDCK cell lines. Expression vectors for such cells ordinarilyinclude (if necessary) an origin of replication, a promoter locatedupstream of the gene to be expressed, along with any necessary ribosomebinding sites, RNA splice sites, polyadenylation site, andtranscriptional terminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often derived from viral material. For example, commonlyused promoters are derived from polyoma, Adenovinrus 2, Cytomegalovirusand most frequently Simian Virus 40 (SV40). The early and late promotersof SV40 virus are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication (Fiers et al., 1978). Smaller or larger SV40 fragmentscan also be used, provided there is included the approximately 250 bpsequence extending from the HindIII site toward the BglI site located inthe viral origin of replication. Further, it is also possible, and oftendesirable, to utilize promoter or control sequences normally associatedwith the desired gene sequence, provided such control sequences arecompatible with the host cell systems.

An origin of replication can be provided with by construction of thevector to include an exogenous origin, such as can be derived from SV40or other viral (e.g., Polyoma, Adeno, VSV, BPV, CMV) source, or can beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

V. Preparing a Recombinant Mu Opioid Receptor Polypeptide orTranscription Regulatory Polypeptide

In yet another embodiment, the present invention contemplates a processof preparing a mu opioid receptor polypeptide comprising transfectingcells with a polynucleotide that encodes a mu opioid receptorpolypeptide to produce transformed host cells; and maintaining thetransformed host cells under biological conditions sufficient forexpression of the polypeptide. Preferably, the transformed host cellsare eukaryotic cells. More preferably still, the eukaryotic cells areCOS cells. Alternatively, the host cells are prokaryotic cells. Morepreferably, the prokaryotic cells are bacterial cells of the DH5α strainof Escherichia coli. Even more preferably, the polynucleotidetransfected into the transformed cells comprise the nucleotide basesequence of SEQ ID NO:1. Most preferably, transfection is accomplishedusing a hereinbefore disclosed expression vector.

In yet another embodiment, the present invention contemplates a processof preparing a gene transcript comprising transfecting cells with apolynucleotide that encodes a gene transcription regulatory polypeptideto produce transformed host cells; and maintaining the transformed hostcells under biological conditions sufficient for expression of thepolypeptide. Preferably, the transformed host cells are eukaryoticcells. More preferably still, the eukaryotic cells are COS cells.Alternatively, the host cells are prokaryotic cells. More preferably,the prokaryotic cells are bacterial cells of the DH5α strain ofEscherichia coli. Even more preferably, the polynucleotide transfectedinto the transformed cells comprise the nucleotide base sequence of SEQID NO:3. Most preferably transfection is accomplished using ahereinbefore disclosed expression vector.

A host cell used in the process is capable of expressing a functional,recombinant mu opioid receptor polypeptide. A preferred host cell is aChinese hamster ovary cell. However, a variety of cells are amenable toa process of the invention, for instance, yeasts cells, human celllines, and other eukaryotic cell lines known well to those of the art.

Following transfection, the cell is maintained under culture conditionsfor a period of time sufficient for expression of a mu opioid receptorpolypeptide. Culture conditions are well known in the art and includeionic composition and concentration, temperature, pH and the like.Typically, transfected cells are maintained under culture conditions ina culture medium Suitable medium for various cell types are well knownin the art. In a preferred embodiment, temperature is from about 20° C.to about 50° C., more preferably from about 30° C. to about 40° C. and,even more preferably about 37° C.

pH is preferably from about a value of 6.0 to a value of about 8.0, morepreferably from about a value of about 6.8 to a value of about 7.8 and,most preferably about 7.4. Osmolality is preferably from about 200milliosmols per liter (mosm/L) to about 400 mosm/l and, more preferablyfrom about 290 mosm/L to about 310 mosm/L. Other biological conditionsneeded for transfection and expression of an encoded protein are wellknown in the art.

Transfected cells are maintained for a period of time sufficient forexpression of a mu opioid receptor polypeptide. A suitable time dependsinter alia upon the cell type used and is readily determinable by askilled artisan. Typically, maintenance time is from about 2 to about 14days.

A recombinant mu opioid receptor polypeptide or gene transcriptionregulatory polypeptide is recovered or collected either from thetransfected cells or the medium in which those cells are cultured.Recovery comprises isolating and purifying the recombinant polypeptide.Isolation and purification techniques for polypeptides are well known inthe art and include such procedures as precipitation, filtration,chromatography, electrophoresis and the like.

VI. Antibodies

In still another embodiment, the present invention provides antibodiesimmunoreactive with a polypeptide of the present invention. Preferably,the antibodies of the invention are monoclonal antibodies. Morepreferably, the polypeptide comprises the amino acid residue sequence ofSEQ ID NO:2 or SEQ ID NO:4. Means for preparing and characterizingantibodies are well known in the art (See, eg., Harlow E. and D. Lane,1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide or polynucleotide of the presentinvention, and collecting antisera from that immunized animal. A widerange of animal species can be used for the production of antiseraTypically an animal used for production of anti-antisera is a rabbit, amouse, a rat, a hamster or a guinea pig. Because of the relatively largeblood volume of rabbits, a rabbit is a preferred choice for productionof polyclonal antibodies.

As is well known in the art, a given polypeptide or polynucleotide mayvary in its immunogenicity. It is often necessary therefore to couplethe immunogen (e.g., a polypeptide or polynucleotide) of the presentinvention) with a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers.

Means for conjugating a polypeptide or a polynucleotide to a carrierprotein are well known in the art and include glutaraldehyde, Mmaleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, immunogencity to a particularimmunogen can be enhanced by the use of non-specific stimulators of theimmune response known as adjuvants. Exemplary and preferred adjuvantsinclude complete Freund's adjuvant, incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

The amount of immunogen used of the production of polyclonal antibodiesvaries inter alia, upon the nature of the immunogen as well as theanimal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal. The production of polyclonal antibodiesis monitored by sampling blood of the immunized animal at various pointsfollowing immunization. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored.

In another aspect, the present invention contemplates a process ofproducing an antibody immunoreactive with a mu opioid receptorpolypeptide comprising the steps of (a) transfecting recombinant hostcells with polynucleotide that encodes a mu opioid receptor polypeptide;(b) culturing the host cells under conditions sufficient for expressionof the polypeptide; (c) recovering the polypeptide; and (d) preparingthe antibodies to the polypeptide. Preferably, the host cell istransfected with the polynucleotide of SEQ ID NO:1 or SEQ ID NO:3. Evenmore preferably, the present invention provides antibodies preparedaccording to the process described above.

A monoclonal antibody of the present invention can be readily preparedthrough use of well-known techniques such as those exemplified in U.S.Pat. No 4,196,265, herein incorporated by reference. Typically, atechnique involves first immunizing a suitable animal with a selectedantigen (e.g., a polypeptide or polynucleotide of the present invention)in a manner sufficient to provide an immune response. Rodents such asmice and rats are preferred animals. Spleen cells from the immunizedanimal are then fused with cells of an immortal myeloma cell. Where theimmunized animal is a mouse, a preferred myeloma cell is a murine NS-1myeloma cell.

The fused spleen/myeloma cells are cultured in a selective medium toselect fused spleen/myeloma cells from the parental cells. Fused cellsare separated from the mixture of non-fused parental cells, for example,by the addition of agents that block the de novo synthesis ofnucleotides in the tissue culture media Exemplary and preferred agentsare aminopterin, methotrexate, and azaserine. Aminopterin andmethotrexate block de novo synthesis of both purines and pyrimidines,whereas azaserine blocks only purine synthesis. Where aminopterin ormethotrexate is used, the media is supplemented with hypoxanthine andthymidine as a source of nucleotides. Where azaserine is used, the mediais supplemented with hypoxanthine.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microliter plates,followed by testing the individual clonal supernatants for reactivitywith an antigen-polypeptides. The selected clones can then be propagatedindefinitely to provide the monoclonal antibody.

By way of specific example, to produce an antibody of the presentinvention, mice are injected intraperitoneally with between about 1-200μg of an antigen comprising a polypeptide of the present invention. Blymphocyte cells are stimulated to grow by injecting the antigen inassociation with an adjuvant such as complete Freund's adjuvant (anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis). At some time (e.g., at least two weeks)after the first injection, mice are boosted by injection with a seconddose of the antigen mixed with incomplete Freund's adjuvant.

A few weeks after the second injection, mice are tail bled and the seratitered by immunoprecipitation against radiolabeled antigen. Preferably,the process of boosting and titering is repeated until a suitable titeris achieved. The spleen of the mouse with the highest titer is removedand the spleen lymphocytes are obtained by homogenizing the spleen witha syringe. Typically, a spleen from an immunized mouse containsapproximately 5×10⁷ to 2×10⁸ lymphocytes.

Mutant lymphocyte cells known as myeloma cells are obtained fromlaboratory animals in which such cells have been induced to grow by avariety of well-known methods. Myeloma cells lack the salvage pathway ofnucleotide biosynthesis. Because myeloma cells are tumor cells, they canbe propagated indefinitely in tissue culture, and are thus denominatedimmortal. Numerous cultured cell lines of myeloma cells from mice andrats, such as murine NS-1 myeloma cells, have been established.

Myeloma cells are combined under conditions appropriate to foster fusionwith the normal antibody-producing cells from the spleen of the mouse orrat injected with the antigen/polypeptide of the present invention.Fusion conditions include, for example, the presence of polyethyleneglycol. The resulting fused cells are hybridoma cells. Like myelomacells, hybridoma cells grow indefinitely in culture.

Hybridoma cells are separated from unfused myeloma cells by culturing ina selection medium such as HAT media (hypoxanthine, aminopterin,thymidine). Unfused myeloma cells lack the enzymes necessary tosynthesize nucleotides from the salvage pathway because they are killedin the presence of aminopterin, methotrexate, or azaserine. Unfusedlymphocytes also do not continue to grow in tissue culture. Thus, onlycells that have successfully fused (hybridoma cells) can grow in theselection media

Each of the surviving hybridoma cells produces a single antibody. Thesecells are then screened for the production of the specific antibodyimmunoreactive with an antigen/polypeptide of the present invention.Single cell hybridomas are isolated by limiting dilutions of thehybridomas. The bybridomas are serially diluted many times and, afterthe dilutions are allowed to grow, the supernatant is tested for thepresence of the monoclonal antibody. The clones producing that antibodyare then cultured in large amounts to produce an antibody of the presentinvention in convenient quantity.

By use of a monoclonal antibody of the present invention, specificpolypeptides and polynucleotide of the invention can be recognized asantigens, and thus identified. Once identified, those polypeptides andpolynucleotide can be isolated and purified by techniques such asantibody-affinity chromatography. In antibody-affinity chromatography, amonoclonal antibody is bound to a solid substrate and exposed to asolution containing the desired antigen. The antigen is removed from thesolution through an immunospecific reaction with the bound antibody. Thepolypeptide or polynucleotide is then easily removed from the substrateand purified.

VII. Pharmaceutical Compositions

In a preferred embodiment, the present invention provides pharmaceuticalcompositions comprising a mu opioid receptor polypeptide or a genetranscription regulatory polypeptide and a physiologically acceptablecarrier. More preferably, a pharmaceutical composition comprises a muopioid receptor polypeptide having the amino acid residue sequence ofSEQ ID NO:2 or SEQ ID NO:4. Even more preferably, a pharmaceuticalcomposition of the invention comprises a polynucleotide that encodes amu opioid receptor polypeptide and a physiologically acceptable carrier.Still more preferably, a pharmaceutical composition of the presentinvention comprises the amino acid residue sequence of SEQ ID NO:2 orSEQ ID NO:4. Alternatively, a pharmaceutical composition comprises thenucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.

A composition of the present invention is typically administeredparenterally in dosage unit formulations containing standard, well-knownnontoxic physiologically acceptable carriers, adjuvants, and vehicles asdesired. The term parenteral as used herein includes intravenous,intramuscular, intraarterial injection, or infusion techniques.

Injectable preparations, for example sterile injectable aqueous oroleaginous suspensions, are formulated according to the known art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a sterile injectable solution orsuspension in a nontoxic parenterally acceptable diluent or solvent, forexample, as a solution in 1,3-butanediol.

Among the acceptable vehicles and solvents that may be employed arewater, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or di-glycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.

Preferred carriers include neutral saline solutions buffered withphosphate, lactate, Tris, and the like. Of course, one purifies thevector sufficiently to render it essentially free of undesirablecontaminants, such as defective interfering adenovirus particles orendotoxins and other pyrogens such that it does not cause any untowardreactions in the individual receiving the vector construct. A preferredmeans of purifying the vector involves the use of buoyant densitygradients, such as cesium chloride gradient centrifugation.

A carrier can also be a liposome. Means for using liposomes as deliveryvehicles are well known in the art [See, e.g. Gabizon, et al., 1990;Ferruti, et al., 1986; and Ranade, 1989].

A transfected cell can also serve as a carrier. By way of example, aliver cell can be removed from an organism, transfected with apolynucleotide of the present invention using methods set forth aboveand then the transfected cell returned to the organism (e.g. injectedintravascularly).

VIII. A Process of Detecting Polynucleotide and the Polypeptides Encoded

Alternatively, the present invention provides a process of detecting apolypeptide of the present invention, wherein the process comprisesimmunoreacting the polypeptide with antibodies prepared according to aprocess described above to form an antibody-polypeptide conjugate anddetecting the conjugates.

In yet another embodiment, the present invention contemplates a processof detecting a messenger RNA transcript that encodes a mu opioidreceptor polypeptide or a gene transcription regulatory polypeptide,wherein the process comprises (a) hybridizing the messenger RNAtranscript with a polynucleotide sequence that encodes the polypeptideto form a duplex; and (b) detecting the duplex. Alternatively, thepresent invention provides a process of detecting a DNA molecule thatencodes a mu opioid receptor polypeptide, wherein the process comprises(a) hybridizing DNA molecules with a polynucleotide that encodes a muopioid receptor polypeptide to form a duplex; and (b) detecting theduplex.

IX. Screening Assays

In yet another aspect, the present invention contemplates a process ofscreening substances for their ability to interact with a mu opioidreceptor polypeptide or a gene transcription regulatory polypeptide, theprocess comprising the steps of providing a polypeptide of the presentinvention and testing the ability of selected substances to interactwith that polypeptide.

Utilizing the methods and compositions of the present invention,screening assays for the testing of candidate substances such asagonists and antagonists of mu opioid receptors can be derived. Acandidate substance is a substance which can interact with or modulate,by binding or other intramolecular interaction, a mu opioid receptorpolypeptide or a gene transcription regulatory polypeptide. In someinstances, such a candidate substance is an agonist of the receptor andin other instances can exhibit antagonistic attributes when interactingwith the receptor polypeptide. In other instances, such substances havemixed agonistic and antagonistic properties or can modulate the receptorin other ways. Alternatively, such substances can promote or inhibittranscription of a mu opioid receptor.

Recombinant receptor expression systems of the present invention possessdefinite advantages over tissue-based systems. The methods of thepresent invention make it possible to produce large quantities of muopioid receptors for use in screening assays. More important, however,is the relative purity of the receptor polypeptides provided by thepresent invention. A relatively pure polypeptide preparation forassaying a protein-protein interaction makes it possible to use elutivemethods without invoking competing, and unwanted, side-reactions.

Cloned expression systems such as those of the present invention arealso useful where there is difficulty in obtaining tissue thatsatisfactorily expresses a particular receptor. Cost is another veryreal advantage, at least with regard to the microbial expression systemsof the present invention. For antagonists in a primary screen,microorganism expression systems of the present invention areinexpensive in comparison to prior art tissue-screening methods.

Traditionally, screening assays employed the use of crude receptorpreparations. Typically, animal tissue slices thought to be rich in thereceptor of interest were the source of the receptor. Alternatively,investigators homogenized the tissue and used the crude homogenate as areceptor source. A major difficulty with this approach is that there areno tissue types where only one receptor type is expressed. The dataobtained therefore could not be definitively correlated with aparticular receptor. With the recent cloning of receptor sub-types andsub-sub-types, this difficulty is highlighted. A second fundamentaldifficulty with the traditional approach is the unavailability of humantissue for screening potential drugs. The traditional approach almostinvariably utilized animal receptors. With the cloning of humanreceptors, there is a need for screening assays which utilize humanreceptors.

With the availability of cloned receptors, recombinant receptorscreening systems have several advantages over tissue based systems. Amajor advantage is that the investigator can now control the type ofreceptor that is utilized in a screening assay. Specific receptorsub-types and sub-sub-types can be preferentially expressed and itsinteraction with a ligand can be identified. Other advantages includethe availability of large amounts of receptor, the availability of rarereceptors previously unavailable in tissue samples, and the lack ofexpenses associated with the maintenance of live animals.

Screening assays of the present invention generally involve determiningthe ability of a candidate substance to bind to the receptor and toaffect the activity of the receptor, such as the screening of candidatesubstances to identify those that inhibit or otherwise modify thereceptor's function. Typically, this method includes preparingrecombinant receptor polypeptide, followed by testing the recombinantpolypeptide or cells expressing the polypeptide with a candidatesubstance to determine the ability of the substance to affect itsphysiological function. In preferred embodiments, the invention relatesto the screening of candidate substances to identify those that affectthe enzymatic activity of the human receptor, and thus can be suitablefor use in humans.

As is well known in the art, a screening assay provides a receptor underconditions suitable for the binding of an agent to the receptor. Theseconditions include but are not limited to pH, temperature, tonicity, thepresence of relevant co-factors, and relevant modifications to thepolypeptide such as glycosylation or prenylation. It is contemplatedthat the receptor can be expressed and utilized in a prokaxyotic oreukaryotic cell. The host cell expressing the receptor can be used wholeor the receptor can be isolated from the host cell. The receptor can bemembrane bound in the membrane of the host cell or it can be free in thecytosol of the host cell The host cell can also be fractionated intosub-cellular fractions where the receptor can be found. For example,cells expressing the receptor can be fractionated into the nuclei, theendoplasmic reticulum, vesicles, or the membrane surfaces of the cell.

pH is preferably from about a value of 6.0 to a value of about 8.0, morepreferably from about a value of about 6.8 to a value of about 7.8 and,most preferably about 7.4. In a preferred embodiment, temperature isfrom about 20° C. to about 50° C., more preferably from about 30° C. toabout 40° C. and, even more preferably about 37° C. Osmolality ispreferably from about 5 milliosmols per liter (mosm/L) to about 400mosm/L and, more preferably from about 200 milliosmols per liter toabout 400 mosm/L and, even more preferably from about 290 mosm/L toabout 310 mosm/L. The presence of co-factors can be required for theproper functioning of the receptor. Typical co-factors include sodium,potassium, calcium, magnesium, and chloride. In addition, small,non-peptide molecules, known as prosthetic groups can be required. Otherbiological conditions needed for receptor function are well known in theart.

It is well known in the art that proteins can be reconstituted inartificial membranes, vesicles or liposomes. (Danboldt et al., 1990).The present invention contemplates that the receptor can be incorporatedinto artificial membranes, vesicles or liposomes. The reconstitutedreceptor can be utilized in screening assays.

It is further contemplated that the receptor of the present inventioncan be coupled to a solid support. The solid support can be agarosebeads, polyacrylamide beads, polyacrylic beads or other solid matricescapable of being coupled to proteins. Well known coupling agents includecyanogen bromide, carbonyidiimidazole, tosyl chloride, andglutaraldebyde.

It is further contemplated that secondary polypeptides which canfunction in conjunction with the receptor of the present invention canbe provided. For example, the receptor of the present invention exertsits physiological effects in conjunction with a G-protein and aneffector polypeptide.

In a typical screening assay for identifying candidate substances, oneemploys the same recombinant expression host as the starting source forobtaining the receptor polypeptide, generally prepared in the form of acrude homogenate. Recombinant cells expressing the receptor are washedand homogenized to prepare a crude polypeptide homogenate in a desirablebuffer such as disclosed herein. In a typical assay, an amount ofpolypeptide from the cell homogenate, is placed into a small volume ofan appropriate assay buffer at an appropriate pH. Candidate substances,such as agonists and antagonists, are added to the admixture inconvenient concentrations and the interaction between the candidatesubstance and the receptor polypeptide is monitored.

Where one uses an appropriate known substrate for the receptor, one can,in the foregoing manner, obtain a baseline activity for therecombinantly produced receptor. Then, to test for inhibitors ormodifiers of the receptor function, one can incorporate into theadmixture a candidate substance whose effect on the receptor is unknown.By comparing reactions which are carried out in the presence or absenceof the candidate substance, one can then obtain information regardingthe effect of the candidate substance on the normal function of thereceptor.

Accordingly, it is proposed that this aspect of the present inventionprovides those of skill in the art with methodology that allows for theidentification of candidate substances having the ability to modify theaction of opioid receptor polypeptides in one or more manners.

In one embodiment, such an assay is designed to be capable ofdiscriminating those candidate substances with the desirable propertiesof opioids but which lack the undesirable properties of opioids. Inanother embodiment, screening assays for testing candidate substancessuch as agonists and antagonists of mu opioid receptors are used toidentify such candidate substances having selective ability to interactwith one or more of the opioid receptor polypeptides but whichpolypeptides are without a substantially overlapping activity with otheropioid receptors.

Additionally, screening assays for the testing of candidate substancesare designed to allow the investigation of structure activityrelationships of opioids with the mu receptors, e.g., study of bindingof naturally occurring hormones or other substances capable ofinteracting or otherwise modulating with the mu receptor versus studiesof the activity caused by the binding of such molecules to the mureceptor. In certain embodiments, the polypeptides of the invention arecrystallized in order to carry out x-ray crystallographic studies as ameans of evaluating interactions with candidate substances or othermolecules with the opioid receptor polypeptide. For instance, thepurified recombinant polypeptides of the invention, when crystallized ina suitable form, are amenable to detection of intra-molecularinteractions by x-ray crystallography.

An important aspect of the invention is the use of recombinantlyproduced mu opioid receptor polypeptide in screening assays for theidentification of substances which can inhibit or otherwise modify oralter the function of the receptor. The use of recombinantly producedreceptor is of particular benefit because the naturally occurringreceptor is present in only small quantities and has proven difficult topurify. Moreover, this provides a ready source of receptor, which hasheretofore been unavailable.

As described above, receptors in the presence of agonists can exerttheir physiological effects through a secondary molecule. A screeningassay of the invention, in preferred embodiments, conveniently employs amu opioid receptor polypeptide directly from the recombinant host inwhich it is produced. This is achieved most preferably by simplyexpressing the selected polypeptide within the recombinant host,typically a eukaryotic host, followed by preparing a crude homogenatewhich includes the polypeptide. A portion of the crude homogenate isthen admixed with an appropriate effector of the mu receptor along withthe candidate substance to be tested. By comparing the binding of theselected effector to the receptor in the presence or absence of thecandidate substance, one can obtain information regarding thephysiological properties of the candidate substance.

The receptor can be expressed in a prokaryotic or a eukaryotic cell.Receptors have been expressed in E. coli (Bertin et al., 1992), in yeast(King et al., (1990) and in mammalian cells (Bouvier et. al. 1988).

A cell expressing a receptor can be used whole to screen agents. Forexample, cells expressing the receptor of the present invention can beexposed to radiolabelled agent and the amount of binding of theradiolabelled agent to the cell can be determined.

The cell expressing the receptor can be fractionated into sub-cellularcomponents which contain the receptor of the present invention. Methodsfor purifying sub-cellular fractions are well known in the art.Sub-cellular fractions include but are not limited to the cytoplasm,cellular membrane, other membranous fractions such as the endoplasmicreticulum, golgi bodies, vesicles and the nucleus. Receptors isolated assub-cellular fractions can be associated with cellular membranes. Forexample, if cellular membrane vesicles are isolated from the cellexpressing the receptor, the receptor molecule can be membrane bound. Itis further contemplated that the receptor of the present invention canbe purified from a cell that expresses the receptor. Methods ofpurification are well known in the art. The purified receptor can beused in screening assays.

In that most such screening assays in accordance with the invention aredesigned to identify agents useful in mimicking the desirable aspects ofopioids while eliminating the undesirable aspects of the hormone,preferred assays employ opioids as the normal agonist.

There are believed to be a wide variety of embodiments that can beemployed to determine the effect of the candidate substance on a mureceptor polypeptide of the invention, and the invention is not intendedto be limited to any one such method. However, it is generally desirableto employ a system wherein one can measure the ability of the receptorpolypeptide to bind to and or be modified by the effector employed inthe presence of a particular substance.

The detection of an interaction between an agent and a receptor can beaccomplished through techniques well known in the art. These techniquesinclude but are not limited to centrifugation, chromatography,electrophoresis and spectroscopy. The use of isotopically labelledreagents in conjunction with these techniques or alone is alsocontemplated. Commonly used radioactive isotopes include ³H, ¹⁴C, ²²Na,³²P, ³⁵S, ⁴⁵Ca. ⁶⁰Co, ¹²⁵I, and ¹³¹I. Commonly used stable isotopesinclude ²H, ¹³C, ¹⁵N, ¹⁸O.

For example, if an agent can bind to the receptor of the presentinvention, the binding can be detected by using radiolabeled agent orradiolabeled receptor. Briefly, if radiolabeled agent or radiolabeledreceptor is utilized, the agent-receptor complex can be detected byliquid scintillation or by exposure to X-Ray film.

The interaction of an agent and a receptor can also be detected by theuse of atomic force microscopy (AFM). Three dimensional images ofbiological materials (e.g. proteins, nucleic acids and membranes) underphysiological conditions can be obtained with nanometer resolutionthrough AFM. AFM has been used to image a number of biologicalspecimens. (Edstrom et al.,, 1990; Drake et al., 1989; Butt et al.,1990; Hoh et al., 1991; Weisenhom et al., 1990; Henderson et al., 1992;Hansma et al., 1992; Durbin, S. D. and W. E. Carlson, 1992; and Lal etal., 1993).

AFM operates by measuring the atomic force between the tip of an AFMprobe and the top surface of the sample being imaged. The probe used forAFM is an integral part of a micro-fabricated cantilever, often made ofSi₃N₄. AFM senses height of the sample surface and controls the verticalposition of the sample by tracking the deflection of the cantilever. Theposition of the cantilever is monitored via laser beam reflection offthe cantilever to an optical position sensor. The signal is used in afeedback mechanism to control the height of the sample. This feedbackmechanism allows the AFM to scan over the sample surface at a constantdeflection, hence a constant force. Because the atomic force is afunction of inter-atomic distance, the height position of the proberepresents the sample surface contour. The vertical features of thesample are thus recorded as the probe is moved over the surface in ahorizontal raster scan, and the image of the sample surface can bedisplayed in real time during imaging and analyzed at a later time.

Recently, the cloned nicotinic acetylcholine receptor expressed inXenopus oocytes was imaged by AFM by the present inventor. The AFM imagerevealed that the acetylcholine receptor was roughly 13 nm, traversingthe lipid bilayer and protruding a few nanometers out of the plasmamembrane and into the cytoplasm The AFM image also showed that theacetylcholine receptors clustered together in the lipid bilayer. Theaverage distance between individual receptors in Xenopus oocytes wasroughly 9-11 nm.

The interaction of an agonist or an antagonist with a mu opioid receptorcan be imaged by AFM. The characterization of intermoleculararrangements and interactions, such as ligand-receptor,antibody-receptor, antibody-transcription regulatory peptide can beachieved by AFM.

When an agent modifies the receptor, the modified receptor can also bedetected by differences in mobility between the modified receptor andthe unmodified receptor through the use of chromatography,electrophoresis or centrifugation. When the technique utilized iscentrifugation, differences in mobility are known as the sedimentationcoefficient. The modification can also be detected by differencesbetween the spectroscopic properties of the modified and unmodifiedreceptor. As a specific example, if an agent covalently modifies areceptor, the difference in retention times between modified andunmodified receptor on a high pressure liquid chromatography (HPLC)column can easily be detected.

As a specific example, where an agent covalently modifies a receptor,the spectroscopic differences between modified and unmodified receptorin the nuclear magnetic resonance (NMR) spectra can be detected.Alternatively, one can focus on the agent and detect the differences inthe spectroscopic properties or the difference in mobility between thefree agent and the agent after modification of the receptor.

Where a secondary polypeptide is provided, the agent-receptor-secondarypolypeptide complex or the receptor-secondary polypeptide complex can bedetected. Differences in mobility or differences in spectroscopicproperties as described above can be detected.

It is further contemplated that where a secondary polypeptide isprovided the enzymatic activity of the effector polypeptide can bedetected. For example, many receptors exert physiological effectsthrough the stimulation or inhibition of adenylyl cyclase. The enzymaticactivity of adenylyl cyclase in the presence of an agent can bedetected.

The interaction of an agent and a receptor can be detected by providinga reporter gene. Well known reporter genes include β-galactosidase(β-Gal), chloramphenicol transferase (CAT) and luciferase. The reportergene is expressed by the host and the enzymatic reaction of the reportergene product can be detected.

In preferred assays, an admixture containing the polypeptide, effectorand candidate substance is allowed to incubate for a selected amount oftime, and the resultant incubated mixture subjected to a separationmeans to separate the unbound effector remaining in the admixture fromany effector/receptor complex so produced. Then, one simply measures theamount of each (e.g., versus a control to which no candidate substancehas been added). This measurement can be made at various time pointswhere velocity data is desired. From this, one can determine the abilityof the candidate substance to alter or modify the function of thereceptor.

Numerous techniques are known for separating the effector fromeffector/receptor complex, and all such methods are intended to fallwithin the scope of the invention. Use of thin layer chromatographicmethods (TLC), HPLC, spectrophotometric, gas chromatographic/massspectrophotometric or NMR analyses. It is contemplated that any suchtechnique can be employed so long as it is capable of differentiatingbetween the effector and complex, and can be used to determine enzymaticfunction such as by identifying or quantifying the substrate andproduct.

The effector/receptor complex itself can also be the subject oftechniques such as x-ray crystallography. Where a candidate substancereplaces the opioid molecule as the drug's mode of action, studiesdesigned to monitor the replacement and its effect on the receptor willbe of particular benefit.

A. Screening Assays for mu Opioid Receptor Polypeptides

The present invention provides a process of screening a biologicalsample for the presence of a mu opioid receptor polypeptide. Abiological sample to be screened can be a biological fluid such asextracellular or intracellular fluid or a cell or tissue extract orhomogenate. A biological sample can also be an isolated cell (e.g., inculture) or a collection of cells such as in a tissue sample orhistology sample. A tissue sample can be suspended in a liquid medium orfixed onto a solid support such as a microscope slide.

In accordance with a screening assay process, a biological sample isexposed to an antibody immunoreactive with the mu opioid receptorpolypeptide whose presence is being assayed. Typically, exposure isaccomplished by forming an admixture in a liquid medium that containsboth the antibody and the candidate opioid receptor polypeptide. Eitherthe antibody or the sample with the opioid receptor polypeptide can beaffixed to a solid support (e.g., a column or a microliter plate).

The biological sample is exposed to the antibody under biologicalreaction conditions and for a period of time sufficient forantibody-polypeptide conjugate formation. Biological reaction conditionsinclude ionic composition and concentration, temperature, pH and thelike.

Ionic composition and concentration can range from that of distilledwater to a 2 molal solution of NaCl. Preferably, osmolality is fromabout 100 mosmols/l to about 400 mosmols/l and, more preferably fromabout 200 mosmols/l to about 300 mosmols/l. Temperature preferably isfrom about 4° C. to about 100° C., more preferably from about 15° C. toabout 50° C. and, even more preferably from about 25° C. to about 40° C.pH is preferably from about a value of 4.0 to a value of about 9.0, morepreferably from about a value of 6.5 to a value of about 8.5 and, evenmore preferably from about a value of 7.0 to a value of about 7.5. Theonly limit on biological reaction conditions is that the conditionsselected allow for antibody-polypeptide conjugate formation and that theconditions do not adversely affect either the antibody or the opioidreceptor polypeptide.

Exposure time will vary inter alia with the biological conditions used,the concentration of antibody and polypeptide and the nature of thesample (e.g., fluid or tissue sample). Means for determining exposuretime are well known to one of ordinary skill in the art. Typically,where the sample is fluid and the concentration of polypeptide in thatsample is about 10⁻¹⁰ M, exposure time is from about 10 minutes to about200 minutes.

The presence of mu opioid receptor polypeptide in the sample is detectedby detecting the formation and presence of antibody-mu opioid receptorpolypeptide conjugates. Means for detecting such antibody-antigen (e.g.,receptor polypeptide) conjugates or complexes are well S known in theart and include such procedures as centrifugation, affinitychromatography and the like, binding of a secondary antibody to theantibody-candidate receptor complex.

In one embodiment, detection is accomplished by detecting an indicatoraffixed to the antibody. Exemplary and well known such indicatorsinclude radioactive labels (e.g., ³²P, ¹²⁵I, ¹⁴C), a second antibody oran enzyme such as horse radish peroxidase. Means for affixing indicatorsto antibodies are well known in the art. Commercial kits are available.

B. Screening Assay for Anti-mu Opioid Receptor Antibody

In another aspect, the present invention provides a process of screeninga biological sample for the presence of antibodies immunoreactive with amu opioid receptor polypeptide (i.e., an anti-mu opioid receptorantibody). In accordance with such a process, a biological sample isexposed to a mu opioid receptor polypeptide under biological conditionsand for a period of time sufficient for antibody-polypeptide conjugateformation and the formed conjugates are detected.

C. Screening Assay for a Polynucleotide that Encodes a mu OpioidReceptor Polypeptide

A DNA molecule and, particularly a probe molecule, can be used forhybridizing as oligonucleotide probes to a DNA source suspected ofpossessing a mu opioid receptor polypeptide encoding polynucleotide orgene. The probing is usually accomplished by hybridizing theoligonucleotide to a DNA source suspected of possessing such a receptorgene. In some cases, the probes constitute only a single probe, and inothers, the probes constitute a collection of probes based on a certainamino acid sequence or sequences of the opioid receptor polypeptide andaccount in their diversity for the redundancy inherent in the geneticcode.

A suitable source of DNA for probing in this manner is capable ofexpressing mu opioid receptor polypeptides and can be a genomic libraryof a cell line of interest. Alternatively, a source of DNA can includetotal DNA from the cell line of interest. Once the hybridization processof the invention has identified a candidate DNA segment, one confirmsthat a positive clone h as been obtained by further hybridization,restriction enzyme mapping, sequencing and/or expression and testing.

Alternatively, such DNA molecules can be used in a number of techniquesincluding their use as: (1) diagnostic tools to detect normal andabnormal DNA sequences in DNA derived from patient's cells; (2) meansfor detecting and isolating other members of the opioid receptor familyand related polypeptides from a DNA library potentially containing suchsequences; (3) primers for hybridizing to related sequences for thepurpose of amplifying those sequences; (4) primers for altering thenative opioid receptor DNA sequences; as well as other techniques whichrely on the similarity of the DNA sequences to those of the opioidreceptor DNA segments herein disclosed.

As set forth above, in certain aspects, DNA sequence informationprovided by the invention allows for the preparation of relatively shortDNA (or RNA) sequences (e.g., probes) that specifically hybridize toencoding sequences of the selected opioid receptor gene. In theseaspects, nucleic acid probes of an appropriate length are prepared basedon a consideration of the selected opioid receptor sequence (e.g., asequence such as that shown in SEQ ID NO:1 or SEQ ID NO:3. The abilityof such nucleic acid probes to specifically hybridize to mu opioidreceptor encoding sequences lend them particular utility in a variety ofembodiments. Most importantly, the probes can be used in a variety ofassays for detecting the presence of complementary sequences in a givensample. However, uses are envisioned, including the use of the sequenceinformation for the preparation of mutant species primers, or primersfor use in preparing other genetic constructions.

To provide certain of the advantages in accordance with the invention, apreferred nucleic acid sequence employed for hybridization studies orassays includes probe sequences that are complementary to at least a 14to 40 or so long nucleotide stretch of the mu opioid receptor encodingsequence, such as that shown in SEQ ID NO:1 and SEQ ID NO:3. A size ofat least 14 nucleotides in length helps to ensure that the fragment isof sufficient length to form a duplex molecule that is both stable andselective. Molecules having complementary sequences over stretchesgreater than 14 bases in length are generally preferred, though, toincrease stability and selectivity of the hybrid, and thereby improvethe quality and degree of specific hybrid molecules obtained. One willgenerally prefer to design nucleic acid molecules havinggene-complementary stretches of 14 to 20 nucleotides, or even longerwhere desired. Such fragments can be readily prepared by, for example,directly synthesizing the fragment by chemical means, by application ofnucleic acid reproduction technology, such as the PCR™ technology ofU.S. Pat. No. 4,683,202, herein incorporated by reference, or byintroducing selected sequences into recombinant vectors for recombinantproduction.

Accordingly, a nucleotide sequence of the present invention can be usedfor its ability to selectively form duplex molecules with complementarystretches of the gene. Depending on the application envisioned, oneemploys varying conditions of hybridization to achieve varying degreesof selectivity of the probe toward the target sequence. For applicationsrequiring a high degree of selectivity, one typically employs relativelystringent conditions to form the hybrids. For example, one selectsrelatively low salt and/or high temperature conditions, such as providedby 0.02M-0.15M NaCl at temperatures of 50° C. to 70° C. Such conditionsare particularly selective, and tolerate little, if any, mismatchbetween the probe and the template or target strand.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate opioid receptor codingsequences from related species, functional equivalents, or the like,less stringent hybridization conditions are typically needed to allowformation of the heteroduplex. Under such circumstances, one employsconditions such as 0.15M-0.9M salt, at temperatures ranging from 20° C.to 70° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

In certain embodiments, it is advantageous to employ a nucleic acidsequence of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization A wide variety ofappropriate indicator means are known in the art, including radioactive,enzymatic or other ligands, such as avidin/biotin, which are capable ofgiving a detectable signal. In preferred embodiments, one likely employsan enzyme tag such a urease, alkaline phosphatase or peroxidase, insteadof radioactive or other environmentally undesirable reagents. In thecase of enzyme tags, calorimetric indicator substrates are known whichcan be employed to provide a means visible to the human eye orspectrophotometrically, to identify specific hybridization withcomplementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes describedherein are useful both as reagents in solution hybridization as well asin embodiments employing a solid phase. In embodiments involving a solidphase, the sample containing test DNA (or RNA) is adsorbed or otherwiseaffixed to a selected matrix or surface. This fixed, single-strandednucleic acid is then subjected to specific hybridization with selectedprobes under desired conditions. The selected conditions depend interalia on the particular circumstances based on the particular criteriarequired (depending, for example, on the G+C contents type of targetnucleic acid, source of nucleic acid, size of hybridization probe,etc.). Following washing of the hybridized surface so as to removenon-specifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label.

D. Screening For Agonists and Antagonists

Mu receptors are one of the major subtypes of opioid receptors.Therefore, highly selective mu opioid receptor agonists are clinicallyuseful.

Development of highly selective, clinically useful mu opioid receptoragonists is facilitated by understanding the specific sites within themu receptor necessary for agonist binding. The recent cloning of therodent mu opioid receptor cDNA has opened up the possibility toinvestigate the structural domains of this receptor subtype that areresponsible for its functioning.

X. Assay Kits

In another aspect, the present invention contemplates diagnostic assaykits for detecting the presence of mu opioid receptor polypeptides inbiological samples, where the kits comprise a first container containinga first antibody capable of immunoreacting with mu opioid receptorpolypeptides, with the first antibody present in an amount sufficient toperform at least one assay. Preferably, assay kits of the inventionfurther comprise a second container containing a second antibody thatimmunoreacts with the first antibody. Preferably the antibodies used inthe assay kits of the present invention are monoclonal antibodies. Evenmore preferably, the first antibody is affixed to a solid support. Morepreferably still, the first and second antibodies comprise an indicator,and, preferably, the indicator is a radioactive label or an enzyme.

The present invention also contemplates a diagnostic kit for screeningagents. Such a kit comprises a mu opioid receptor of the presentinvention. The kit can further contain reagents for detecting aninteraction between an agent and a receptor of the present invention.The provided reagent can be radiolabeled. The kit can contain a knownradiolabeled agent capable of binding or interacting with a receptor ofthe present invention.

It is further contemplated that the kit can contain a secondarypolypeptide. The secondary polypeptide can be a G-protein. The secondarypolypeptide can also be an effector protein. When a secondarypolypeptide is included in a kit, reagents for detecting an interactionbetween the receptor and the secondary polypeptide can be provided. As aspecific example, an antibody capable of detecting a receptor/G-proteincomplex can be provided. As another specific example, an antibodycapable of detecting a G-protein/effector complex can be provided.Reagents for the detection of the effector can be provided. For example,if the effector provided is adenylyl cyclase, reagents for detecting theactivity of adenylyl cyclase can be provided. The identity of suchagents is within the knowledge of those skilled in the relevant art.

In an alternative aspect, the present invention provides diagnosticassay kits for detecting the presence, in biological samples, of apolynucleotide that encodes a mu opioid receptor polypeptides, the kitscomprising a first container that contains a second polynucleotideidentical or complementary to a segment of at least 10 contiguousnucleotide bases of SEQ ID NO:1 or SEQ ID NO:3.

In another embodiment, the present invention contemplates diagnosticassay kits for detecting the presence, in a biological sample, ofantibodies immunoreactive with mu opioid receptor polypeptides, the kitscomprising a first container containing a mu opioid receptor polypeptidethat immunoreacts with the antibodies, with the polypeptides present inan amount sufficient to perform at least one assay. The reagents of thekit can be provided as a liquid solution, attached to a solid support oras a dried powder. Preferably, when the reagent is provided in a liquidsolution, the liquid solution is an aqueous solution. Preferably, whenthe reagent provided is attached to a solid support, the solid supportcan be chromatograph media or a microscope slide. When the reagentprovided is a dry powder, the powder can be reconstituted by theaddition of a suitable solvent. The solvent can be provided.

EXAMPLES

Examples are included to illustrate preferred modes of the invention.Certain aspects of the following examples are described in terms oftechniques and procedures found or contemplated by the present inventorsto work well in the practice of the invention. These examples areexemplified through the use of standard laboratory practices of theinventor. In light of the present disclosure and the general level ofskill in the art, those of skill will appreciate that the followingexamples are intended to be exemplary only and that numerous changes,modifications and alterations can be employed without departing from thespirit and scope of the invention.

Example I

Isolation of cDNA Clones

Low stringency hybridization was utilized for isolating opioid receptorsrelated to the mouse δ-opioid receptor (Evan et al., 1992; Kieffer etal., 1992) because all three types of opioid receptors share sequencehomology, share overlapping pharmacology, couple to G proteins, and havea common effect on Ca²⁺ and K⁺ channels (Pastrenak G. W. 1988).Oligodeoxynucleotides were synthesized based on the mouse δ-opioidreceptor sequence (Evan et al., 1992 and Kieffer et al., 1992) and wereused to amplify, by PCR, a sequence fragment from a rat brain cDNAlibrary (Snutch et al., 1990).

Two primers, ATCTTCACCCTCACCATGATG (SEQ. ID. NO:5) andCGGTCCITCTCCTTGGAACC (SEQ. ID. NO:6), were synthesized from the sequenceof the mouse δ-opioid receptor (Evans et al., 1992; Kieffer et al.,1992), corresponding to the third transmembrane domain and the thirdcytoplasmic loop, respectively. PCR™ was performed using purified DNAfrom a rat brain cDNA library (Snutch et al., 1990), in an airThermo-cycler (Idaho Technology) under modified conditions (94° for 10sec, 56° for 20 sec, and 72° for 40 sec, for 40 cycles). A 356 bpfragment was purified and subcloned into pBLUESCRIPT SK(+) vector.Sequence analysis of the resulting 356 bp PCR™ product revealed completeidentity with the corresponding portion of the δ-opioid receptor (Evanset al., 1992), showing a conserved relationship between the δ-opioidreceptors from these two species.

The 356-bp fragment was then used to screen a rat brain cDNA libraryunder low stringency conditions (6×SSPE (1.08M NaCl, 60 mM NaH₂PO₄, 6 mMEDTA, pH 7.4), 5× Denhardt solution, 0.5% sodium dodecyl sulfate, 100μg/ml salmon sperm DNA, at 50°). The final wash was carried out in 0.5×standard saline citrate (7 mM NaCl, 7.5 mM sodium citrate), 0.1% sodiumdodecyl sulfate, at 50° C. Phagemids were rescued from positive λ clonesby infection with helper phage. Two independent isolates were used forsequence determination by shotgun cloning into pBLUESCRIPT SK(+).Subsequent sequencing of both strands from each isolate showed these twoclones to be identical. Potential post-translational modification siteswere identified by using the PCGENE program. Comparison of the sequencewith other receptors was performed by using the BLAST program (NationalInstitutes of Health).

Sequence analysis revealed that one cDNA clone contained an open readingframe of 1194 bp, encoding a protein of 398 amino acids. Hydropathyanalysis of the deduced protein indicated seven hydrophobic domains,typical of G protein-coupled receptors (Collins et al., 1991). Thisprotein, termed MOR-1, shows high levels of homology with the mouseδ-opioid receptor DOR-1 (Evans et al., 1992) (64%) and rat somatostatinreceptors (Meyerhof et al., 1991 and Kluxen et al., 1992) (44%) (FIG.1). MOR-1 also displays moderate homology (30-32%) with several Gprotein-coupled receptors, including the angiotensin II receptor, theinterleukin-8 receptor, the N-formyl peptide receptor, and the C-Cchemokine receptor. The sequence homology is lower (≧25%) between MOR-1and other G protein-coupled receptors, such as the adrenergic andmuscarinic receptors (Collins et al., 1991). At the amino acid sequencelevel MOR-1 contains several sites that are conserved among other Gprotein-coupled receptors (Collins et al., 1991). Aspartic acid residuesthought to interact with the protonated amine group of various ligandsappear in putative transmembrane domains II and III, and two conservedcysteine residues believed to be involved in disulfide bonding occur inthe first and second extracellular loop domains (Dixon et al., 1988).Both of these features are conserved between MOR-1 and the δ-opioidreceptor (FIG. 1). In addition, MOR-1 displays a cysteine residue in thecarboxyl-terminal region that is conserved among many G protein-coupledreceptors which likely serves as a target for palmitoylation (Collins etal., 1991). There are also multiple sites in the second and thirdintracellular loops as well as the carboxyl-terminal region that canundergo phosphorylation via protein kinase A and protein kinase CCompared with the mouse δ-opioid receptor, MOR-1 contains five insteadof two asparagine residues in the amino-terminal region that match theconsensus sequence for N-linked glycosylation. These glycosylation sitesare important in the modulation of receptor expression and function(Sumikawa K. and R. Miledi, 1989). The sequence of the MOR-1 cDNA hasbeen submitted to GenBank (accession number L13069).

Example II

Expression of Rat Mu-Opioid Receptor

A 1.4-kilobase HindIII fragment encompassing the open reading frame fromthe cDNA encoding MOR-1 was cloned downstream of the humancytomegalovirus promoter in the mammalian expression vector pRc/CMV(Invitrogen). COS-7 cells grown in Dulbecco's modified Eagle's medium(Sigma D-5648) supplemented with 10% fetal bovine serum and 2 mMglutamine were transfected with supercoiled DNA by eitherelectroporation or CaPO₄ co-precipitation (Graham, F. L. and A. J. VanDer Eb, 1973). Electroporation was performed in 0.4 cm cuvettes at 200V, using 3×10⁶ cells in a total volume of 0.5 ml containing growthmedium, 40 μg of expression plasmid, and 200 μg of sheared salmon spermDNA. Cells were harvested 48-72 hr after electroporation transfection.

The plasmid was transiently transfected into COS-7 cells to expressMOR-1, and membranes from these cells were prepared. Cells wereharvested by scraping into phosphate-buffered saline, pH 7.2, andcentrifuged. Cell pellets were resuspended in lysis buffer (20 mMTris-HCl, pH 7.4, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) andlysed with a Dounce homogenizer fitted with a tight pestle. Thesuspension was centrifuged for 10 min at 1000×g, and the supernatant wasremoved to a fresh tube. The pellet was resuspended in lysis buffer andcentrifuged as described above. The supernatants were then combined andcentrifuged for 20 min at 35,000×g. Membranes were washed in 50 mMTris-HCl, PH 7.4, and centrifuged for 20 min at 35,000×g. The membranepellets were then suspended in 50 mM Tris-HCl, pH 7.4. Proteinconcentrations were determined by the method of Bradford (Bradford, M.M., 1976).

Binding studies of membrane aliquots (15-50 μg/reaction) from thetransfected COS-7 cells were carried out in 50 mM Tris-HCl, pH 7.4, 0.2%bovine serum albumin, at 4° for 90 min. A range of 0.01-2.5 nM[³H]diprenorphine was used in the saturation assay and 0.25 nM was usedfor the displacement experiment The reactions were terminated by vacuumfiltration through Whatman GF/B filters which were pretreated with 1%polyethylenimine. Nonspecific binding was determined using 5 μMnaloxone.

Saturation binding of membranes was performed using [³H]diprenorphine(Magnan et al., 1982), a nonselective opioid antagonist with highaffinity for all three types of opioid receptors FIG. 2. Membranes ofCOS cells transfected with the MOR-1 plasmid displayed [³H]diprenorphinebinding with a dissociation constant (K_(d)) value of 0.3±0.09 nM(mean±standard error, five experiments). This is one tenth the K_(d)value (3.8 nm) reported for the cloned mouse δ-opioid receptor (Evans etal., 1992).

Various ligands which displace [³H]diprenorphine binding were used tocharacterize the pharmacological features of MOR-1. The inhibitionconstant K_(i) values were obtained from three binding experiments foreach ligand and are listed in Table 2.

TABLE 2 Ligand K_(i) values (nM) Agonists (DAGO) (DAMGO) 2.8 ± 0.2(DADLE) 55 ± 17 (DSLET) 314 ± 35 U-50488 1,551 ± 307 (DPDPE) 7,297 ±1,092 Antagonists and Somatostatins Naloxone 1.0 ± 0.6 β-funaltrexamine(β-FNA) 1.3 ± 0.1 Naloxonazine 2.4 ± 0.9 Cyprodime 9.1 ± 2.8 Cyclicsomatostatin 10,994 ± 6,777 (IC₅₀) Somatostatin-1-14 730,000 (IC₅₀)

The μ-selective agonist [D-Ala², N—Me'Phe⁴, Gly—ol⁵]-enkephalin (DAGO)displaced diprenorphine binding with high affinity (K_(i)=2.8 nM),whereas the δ-selective agonist [D-Pen²⁵]-enkephalin (DPDPE) and theκ-selective agonist U-50488 showed low affinities, with K_(i) values inthe micromolar range (Pasternak et al.,, 1980). [D-Ala²,D-Leu⁵]-enkephalin (DADLE) and [D-Ser², Leu⁵, Thr⁶]-enkephalin (DSLET),two predominantly δ agonists that have previously been shown to interactwith μ receptors (Barrett, R. W. and J. L Vaught, 1983; Itzhak, Y. andG. W. Pasternak, 1987), showed binding to MOR-1 with moderate affinities(K_(i)=55 and 314 nM, respectively). The rank order of potency for theseopioid agonists is DAGO>DADLE>DSLET>U-50488>DPDPE which is thepharmacological profile of μ receptors. Displacement of diprenorphinebinding to MOR-1 was performed with three μ-selective antagonists,β-FNA, naloxonazine, and cyprodime (Ward et al., 1985; Nishimura et al.,1984; Curciani et al., 1987; Schmidhammer et al., 1990). All threeligands exhibited high potency in displacing diprenorphine binding toMOR-1 with K_(i) values in the nanomolar range (Table 1). The order ofpotency for opioid agonists and the nanomolar affinity for μ-selectiveantagonists indicates that MOR-1 is a μ-opioid receptor.

The sequence homology between rat μ-opioid receptor encoded by MOR-1cDNA and the somatostatin receptors is noteworthy. Many somatostatinanalogues, especially those of the cyclic form, have been shown tointeract with μ-opioid receptors, and some of them have been used asμ-selective antagonists (Gulya et al., 1986). Displacement bindingexperiments were performed using two somatostatin ligands,somatostatin-1-14 and cyclic somatostatin. Somatostatin-1-14 did notdisplace [³H]diprenorphine binding to the rat μ receptor encoded by theMOR-1 cDNA, at concentrations as high as 30 μM, whereas cyclicsomatostatin competed with diprenorphine binding with an IC₅₀ value inthe micromolar range FIG. 3A and FIG. 3B; Table 1).

All three classes of opioid receptors are coupled to adenylyl cyclase(Childers S. R, 1993; Cox, B. M., 1993; Sharma et al., 1975). cAMPlevels were determined in COS-7 cells after exposure to μ-selectiveligands to examine whether the μ receptor is coupled to intracellularsignaling pathways. COS-7 cells transiently expressing the MOR-1 plasmidcDNA were harvested 48 hrs after transfection and were resuspended ingrowth medium. Cells were treated with 10 μM forskolin in the presenceof 1 mM 3-isobutyl-1-methylxanthine at 37° for 10 min. DAGO (100 nM) andnaloxonazine (10 μM) were included during forskolin treatment whereindicated. Cells were pelleted and then solubilized in 0.1 N HCl. Afterextraction with water-saturated ether, the supernatants werelyophilized. cAMP was assayed using a commercially availableradioimmunoassay kit (DuPont/NEN). Results are shown in FIG. 14. Innontransfected COS-7 cells, treatment with these ligands did not causesignificant changes in the intracellular cAMP levels. In transfectedcells expressing the μ receptor, the μ-specific agonist DAGO reducedcAMP levels significantly (18.1±25% reduction from control, p<0.05).This inhibitory effect on adenylyl cyclase activity by DAGO was blockedby the μ-selective antagonist naloxonazine. It has been reported thatμ-opioid receptors exert an inhibitory effect on adenylyl cyclaseactivity (Frey, E. A. and J. W. Kebabian, 1984) and that activation of μreceptors in a human neuroblastoma cell line reduces intracellular cAMPlevels by approximately 20% (Yu et al., 1986). Our data are consistentwith these reports and shows that the μ-opioid receptor encoded by MOR-1is functionally coupled to the inhibition of adenylyl cyclase.

Example III

Stable Transfection of Mammalian Cells

Stably transfected cultured mammalian cells were generated bytransfecting chinese hamster ovary cells (CHO) with the vector pRc/CMVwhich contained a cDNA coding for a mu opioid receptor. CHO cells fromthe American Type Culture Collection were plated at a density of 5×10⁴cells/100 mm plate one day before transfection. The CHO cells wereincubated at 37° C. in a humidified chamber with 5% CO₂, in DME (Sigma),supplemented with 2 mM glutamine and 10% fetal bovine serum. The culturemedium was changed 4-6 hours before transfection. The plasmid DNA wastransfected into cells using the calcium phosphate precipitation method(Graham and Van Der Eb, 1973). After the CaPO₄ precipitation, the plateswere incubated in a 3% CO₂, humidified chamber at 37° C. After 15-24 hrat 3% CO₂, the cells were washed with Hank's balanced salt solution,fresh culture medium was added, and the cells were transferred to a 5%CO₂, humidified incubator at 37° C. After 24 hr at 5% CO₂, selection forneomycin resistance was initiated by replacing the culture medium withmedium containing 500 μg/ml of G418 (Sigma). G418 is a neomycin analoguethat is permeable to mammalian cell membranes. The selection medium waschanged every 2-3 days until drug-resistant colonies formed (2-3 weeks).Individual colonies were picked and replated after trypsin dissociation.A second round of selection was performed to isolate clonal derivatives.G418 resistant colonies were then allowed to grow in G418 medium untilthe plates were confluent. Aliquots of cells G418 resistant cells werefrozen in liquid nitrogen for long-term storage of the transfectedcells.

The expression of the mu opioid receptor in stably transfected CHO cellswas demonstrated by saturation binding studies using a range of 0.2-20mM ³H-DAGO. Membranes from G418 resistant CHO cells were prepared asdescribed in Example II above. The B_(MAX) of ³H-DAGO binding tomembranes from stably transfected CHO cells was 660 nmole/mg protein,and the K_(D) is ˜1 nM for ³H-DAGO. These values are comparable to thevalues obtained for ³H-DAGO binding to mu opioid receptors obtained fromtransient transfections of COS-7 cells.

As expected for stable transfectants, different clonal derivatives gavedifferent levels of receptor expression. For example, clones #15 and #18had 1 and 3 picomoles of the receptor per milligrams of membraneprotein, respectively as determined by ligand binding.

Functional characteristics of the expressed mu opioid receptor in stabletransfection were also determined by assaying the GTPase activity of Gproteins (Koshi and Klee, 1981). Upon stimulation by 10 μM DAMGO, theGTPase activity was increased by 30%, indicating that the G proteinswere activated by the mu opioid receptor. This effect of DAMGO wasblocked by naloxone.

REFERENCES CITED

The references listed below as well as all references cited in thespecification are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for or teach methodology,techniques and/or compositions employed herein.

Adelman et al. (1983) DNA 2:183.

Akil, H. et al. (1984) Annu. Rev. Neurosci. 7:223.

Attali, B. et al. (1989) J. Neurochem. 52:360.

Barrett, R. W. and J. L. Vaught (1983) Life Sci. 33:2439.

Berg J. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:99-102.

Bertin, B. et al. (1992) J. Biol. Chem. 267(12):8200.

Bero et al. (1988) Mol. Pharmacol. 34:614.

Bertolucci, M. et al. Neurosci. Abstr. 18L1368.

Bolivar et al. (1977) Gene, 2:95.

Boshart et al. (1985) Cell 41:521.

Bouvier, M. et al. (1988) Mol. Pharmacol. 33:133.

Bradford, M. M. (1976) Anal. Biochem. 72:248.

Bradbury, A. F. et al. (1976) Nature 260:165.

Breder, C. D. et al. (1992). J. Neurosci 12:3920.

Butt et al. (1990) Biophys. J. 58:1473.

Chang et al. (1978) Nature, 375:615.

Childers, S. R. (1993) Handb. Exp. Pharmacol. Sci. 104:189.

Clark, J. A. et al. (1989) J. Pharmacol. Expt. Therapeut. 251:461.

Collins et al. (1991) Vitam. Horm. 46:1.

Corbett et al. (1993) Handb. Exp. Pharmacol. Sci 8:456.

Cotecchia et al. (1988) Proc. Natl. Acad. Sci. USA 85:7159.

Cotecchia et al. (1990) Proc. Natl. Acad. Sci. USA 87:2896.

Cox, B. M. (1993) Handb. Exp. Pharmacol. Sci. 104:145.

Crea et al. (1978) Proc. Natl. Acad. Sci. U.S.A., 75:5765.

Cruciani et al. (1987) J. Pharmacol. Exp. Ther. 24215.

Danboldt, N. C. et al. (1990) Biochemistry 29(28):6734.

DiChiara, G. et al. (1992) Trends Pharmacol. Sci. 13:185.

Dohlman (1987) Biochemistry 26:2657.

Dohlman, H. G. (1991) Annu. Rev. Biochem. 60:166-170; 174-s176; 653-688.

Drake et al. (1989) Science 243:1586.

Durbin, S. D. and W. E. Carlson (1992) J. Crystal Growth 122:71.

Edstrom et al. (1990) Biophys. J. 58:1437.

Evans et al. (1992) Science 258:1952.

Evans, R. M. and S. M. Hollenberg (1988) Cell 52:1-3.

Dixon et al. (1988) Cold Spring Harbor Symp. Quant. Biol. 53:487.

Ferruti, P. and M. C. Tanzi, (1986) Cris. Rev. Ther. Drug Carrier Syst.2:117.

Fiers et al. (1978) Nature 273:113.

Frey, E. A and J. W. Kebabian (1984) Endocrinology 115:1797.

Frielle, T. et al. (1988) Proc. Natl. Acad. Sci. USA 85:9484.

Gabizon, A. et al. (1990) Cancer Res. 50:6371-6378.

Gioannini, T. L. et al. (1989) J. Mol. Recogn. 2:44.

Goeddel et al. (1979) Nature, 281:544.

Goeddel et al. (1980) Nucleic Acids Res., 8:4057.

Graham, F. L and A. J. Van Der Eb (1973) Virology 52:456.

Gransch, C. et al. (1988) J. Biol. Chem. 263:5853.

Gulya et al. (1986) Life Sci. 38:2221.

Hansma et al. (1992) Science 256:1180.

Harlow, E. and D. Lane (1988) Antibodies: “A Laboratory Manual,” ColdSpring Harbor Laboratory.

Henderson et al. (1992) Science 257:1944.

Hess et al. (1968) J. Adv. Enzyme Reg. 7:149.

Hitzeman et al. (1980) J. Biol. Chem. 255:2073.

Hob et al (1991) Science 253:1405.

Holland et al. (1978) Biochemistry 17:4900.

Horstman, D. A et al. (1990) J. Biol. Chem. 265:21590.

Hsia, J. A et al. (1984) J. Biol. Chem. 259:1086.

Hughes, J. et al. (1975) Nature 258:577.

Itakura et al. (1977) Science 198:1056.

Itzbak, Y. and G. W. Pasternak (1987) Life Sci. 40:307.

Johnson et al. (1990) Mol. Pharm., 38:289.

Jones (1977) Genetics 85:12.

Kanaho et al. (1984) J. Biol. Chem. 259:7378.

Kennelly, P. J. et al. (1991) J. Biol. Chem. 266:15555.

Kieffer et al. (1992) Proc. Natl. Acad. Sci. USA 89:12048.

King et al. (1990) Science 250:121.

Kingsman et al. (1979) Gene 7:141.

Klug, A. and D. Rhodes (1987) Trends Biochem. Sci. 12:464-469.

Kluxen et al. (1992) Proc. Natl. Acad. Sci. USA 89:4618.

Kobilka, B. K. et al. (1987) J. Biol. Chem. 262:7321.

Kobilka, B. K. et al. (1988) Science 240:1310.

Koob, G. F. et al. (1992) Trends Neurosci. 15:186.

Koshi, G. and W. A. Klee (1981) Proc. Natl. Acad. Sci. USA 78:4185.

Kozasa et al. (1988) Proc. Natl. Acad. Sci USA 85:2081.

Kruse and Patterson, eds. (1973) Tissue Culture, Academic Press.

Kyte, J., and R. F. Doolittle (1982) J. Mol. Biol. 157:105.

Lal et al. (1993) Am. J. Physiol. in press.

Lal, R. and L. Yu (1993) Proc. Natl. Acad. Sci. USA, 90:7280.

Loh, H. H. et al. (1990) Annu. Rev. Pharmacol. Toxicol. 30:123.

Lomasney et al. (1990) Proc. Natl. Acad. Sci. USA 87:5094.

Lutz, R. A et al. (1992) J. Receptor Res. 12:267.

Magnan et al. (1982) Naunyn-Schmiedebergs Arch. Pharmacol. 319:197.

Mansour, A. et al. (1987) J. Neurosci. 7:2445.

Marullo et al. (1988) Proc. Natl. Acad. Sci. USA 85:7551.

Messing et al., Third Cleveland Symposium on Macromolecules andRecombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981).

Meryerhof et al. (1991) DNA Cell Biol. 10:689.

Miller, J. et al. (1985) EMBO J. 4:1609-1614.

Nathans et al. (1986 A) Science 232:193.

Nathans et al. (1986 B) Science 232:203.

Nishimura et al. (1984) Mol. Pharmacol. 25:29.

Nock, B. et al. (1988) Eur. J. Pharmacol. 154:27.

Okayama et al. (1983) Mol. Cell Biol. 3:280.

Olson, G. A. et al. (1989) Peptides 10:1253.

Ott, S. et al. (1988) J. Biol. Chem. 263:10524.

Parker, E. and E. M. Ross (1991) J. of Biol. Chem. 266:15.

Pasternak, G. W. (1988) The Opiate Receptors Humana Press, Clifton, N.J.

Payette et al. (1990) FEBS Lett. 266-21.

Payre, F. and A Vincent (1988) FEBS Lett. 234:245-250.

Pert, C. G. et al. (1973) Science 179:1011.

Pert, C. B. et al. (1974) Mol. Pharmacol. 10:868.

Pfeiffer, A. et al. (1986) Science 223:774.

Puttfarcken, P. S. et al. (1988) Mol. Pharmacol. 33:520.

Ranade, V. V. (1989) J. Clin. Pharmacol. 29:685-694

Regan et al. (1988) Proc. Natl. Acad. Sci. USA 85:6301.

Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Schmidhammer et al. (1990) Prog. Clin. Biol. Res. 328:37.

Seeburg (1982) DNA 1:239.

Sharma et al. (1975) Proc. Natl. Acad. Sci. USA 72:590.

Shook, J. E. et al. (1990) Am. Rev. Respir. Dis. 142:895.

Siebwenlist et al. (1980) Cell, 20:269.

Simon, E. J. (1991) Medicinal Res. Rev. 11:357.

Snutch et al. (1990) Proc. Natl. Acad. Sci. USA 87:3391.

Stinchcomb et al. (1979) Nature, 282:39.

Stratford-Perricaudet et al. (1992).

Strotchman and Simon (1991).

Sumikawa, K and R. Miledi (1989) Mol. Brain Res. 5:183.

Thomsen et al. (1984) PNAS 81:659.

Tschemper et al. (1980) Gene 10:157.

Unterwald, E. M. et al. (1991) Brain Res. 562:57.

Unterwald, E. M. et al. (1987) Eur. J. Pharmacol. 133:275.

Ward et al. (1985) Eur. J. Pharmacol. 107:323.

Weisenhorn et al. (1990) Biophys J. 58:1251.

Xie, G-X. et al. (1992) Proc. Natl. Acad. Sci. USA 89:4124.

Yamada, Y. et al. (1992) Proc. Natl. Acad. Sci. USA 89:251.

Yasuda, K et al. (1992) J. Biol. Chem. 267:20422.

Yokota, Y. et al. (1992) EMBO J. 11:3585.

Yu et al. (1986) J. Biol. Chem. 261:1065.

Zukin, R. S. et al. (1988) Proc. Natl. Acad. Sci. USA 85:4061.

9 1618 base pairs nucleic acid single linear unknown CDS 214..1407 1CGTGGAAGGG GGCTACAAGC AGAGGAGAAT ATCAGACGCT CAGACGTTCC CTTCTGCCTG 60CCGCTCTTCT CTGGTTCCAC TAGGGCTGGT CCATGTAAGA ATCTGACGGA GCCTAGGGCA 120GCTGTGAGAG GAAGAGGCTG GGGCGCGTGG AACCCGAAAA GTCTGAGTGC TCTCAGTTAC 180AGCCTACCTA GTCCGCAGCA GGCCTTCAGC ACC ATG GAC AGC AGC ACC GGC CCA 234 MetAsp Ser Ser Thr Gly Pro 1 5 GGG AAC ACC AGC GAC TGC TCA GAC CCC TTA GCTCAG GCA AGT TGC TCC 282 Gly Asn Thr Ser Asp Cys Ser Asp Pro Leu Ala GlnAla Ser Cys Ser 10 15 20 CCA GCA CCT GGC TCC TGG CTC AAC TTG TCC CAC GTTGAT GGC AAC CAG 330 Pro Ala Pro Gly Ser Trp Leu Asn Leu Ser His Val AspGly Asn Gln 25 30 35 TCC GAT CCA TGC GGT CTG AAC CGC ACC GGG CTT GGC GGGAAC GAC AGC 378 Ser Asp Pro Cys Gly Leu Asn Arg Thr Gly Leu Gly Gly AsnAsp Ser 40 45 50 55 CTG TGC CCT CAG ACC GGC AGC CCT TCC ATG GTC ACA GCCATT ACC ATC 426 Leu Cys Pro Gln Thr Gly Ser Pro Ser Met Val Thr Ala IleThr Ile 60 65 70 ATG GCC CTC TAC TCT ATC GTG TGT GTA GTG GGC CTC TTC GGAAAC TTC 474 Met Ala Leu Tyr Ser Ile Val Cys Val Val Gly Leu Phe Gly AsnPhe 75 80 85 CTG GTC ATG TAT GTG ATT GTA AGA TAC ACC AAA ATG AAG ACT GCCACC 522 Leu Val Met Tyr Val Ile Val Arg Tyr Thr Lys Met Lys Thr Ala Thr90 95 100 AAC ATC TAC ATT TTC AAC CTT GCT CTG GCA GAC GCC TTA GCG ACCAGT 570 Asn Ile Tyr Ile Phe Asn Leu Ala Leu Ala Asp Ala Leu Ala Thr Ser105 110 115 ACA CTG CCC TTT CAG AGT GTC AAC TAC CTG ATG GGA ACA TGG CCCTTC 618 Thr Leu Pro Phe Gln Ser Val Asn Tyr Leu Met Gly Thr Trp Pro Phe120 125 130 135 GGA ACC ATC CTC TGC AAG ATC GTG ATC TCA ATA GAT TAC TACAAC ATG 666 Gly Thr Ile Leu Cys Lys Ile Val Ile Ser Ile Asp Tyr Tyr AsnMet 140 145 150 TTC ACC AGC ATA TTC ACC CTC TGC ACC ATG AGC GTG GAC CGCTAC ATT 714 Phe Thr Ser Ile Phe Thr Leu Cys Thr Met Ser Val Asp Arg TyrIle 155 160 165 GCT GTC TGC CAC CCA GTC AAA GCC CTG GAT TTC CGT ACC CCCCGA AAT 762 Ala Val Cys His Pro Val Lys Ala Leu Asp Phe Arg Thr Pro ArgAsn 170 175 180 GCC AAA ATC GTC AAC GTC TGC AAC TGG ATC CTC TCT TCT GCCATC GGT 810 Ala Lys Ile Val Asn Val Cys Asn Trp Ile Leu Ser Ser Ala IleGly 185 190 195 CTG CCT GTA ATG TTC ATG GCA ACC ACA AAA TAC AGG CAG GGGTCC ATA 858 Leu Pro Val Met Phe Met Ala Thr Thr Lys Tyr Arg Gln Gly SerIle 200 205 210 215 GAT TGC ACC CTC ACG TTC TCC CAC CCA ACC TGG TAC TGGGAG AAC CTG 906 Asp Cys Thr Leu Thr Phe Ser His Pro Thr Trp Tyr Trp GluAsn Leu 220 225 230 CTC AAA ATC TGT GTC TTT ATC TTC GCT TTC ATC ATG CCGATC CTC ATC 954 Leu Lys Ile Cys Val Phe Ile Phe Ala Phe Ile Met Pro IleLeu Ile 235 240 245 ATC ACT GTG TGT TAC GGC CTG ATG ATC TTA CGA CTC AAGAGC GTT CGC 1002 Ile Thr Val Cys Tyr Gly Leu Met Ile Leu Arg Leu Lys SerVal Arg 250 255 260 ATG CTA TCG GGC TCC AAA GAA AAG GAC AGG AAT CTG CGCAGG ATC ACC 1050 Met Leu Ser Gly Ser Lys Glu Lys Asp Arg Asn Leu Arg ArgIle Thr 265 270 275 CGG ATG GTG CTG GTG GTC GTG GCT GTA TTT ATC GTC TGCTGG ACC CCC 1098 Arg Met Val Leu Val Val Val Ala Val Phe Ile Val Cys TrpThr Pro 280 285 290 295 ATC CAC ATC TAC GTC ATC ATC AAA GCG CTG ATC ACGATT CCA GAA ACC 1146 Ile His Ile Tyr Val Ile Ile Lys Ala Leu Ile Thr IlePro Glu Thr 300 305 310 ACA TTT CAG ACC GTT TCC TGG CAC TTC TGC ATT GCTTTG GGT TAC ACG 1194 Thr Phe Gln Thr Val Ser Trp His Phe Cys Ile Ala LeuGly Tyr Thr 315 320 325 AAC AGC TGC CTG AAT CCA GTT CTT TAC GCC TTC CTGGAT GAA AAC TTC 1242 Asn Ser Cys Leu Asn Pro Val Leu Tyr Ala Phe Leu AspGlu Asn Phe 330 335 340 AAG CGA TGC TTC AGA GAG TTC TGC ATC CCA ACC TCGTCC ACG ATC GAA 1290 Lys Arg Cys Phe Arg Glu Phe Cys Ile Pro Thr Ser SerThr Ile Glu 345 350 355 CAG CAA AAC TCC ACT CGA GTC CGT CAG AAC ACT AGGGAA CAT CCC TCC 1338 Gln Gln Asn Ser Thr Arg Val Arg Gln Asn Thr Arg GluHis Pro Ser 360 365 370 375 ACG GCT AAT ACA GTG GAT CGA ACT AAC CAC CAGCTA GAA AAT CTG GAG 1386 Thr Ala Asn Thr Val Asp Arg Thr Asn His Gln LeuGlu Asn Leu Glu 380 385 390 GCA GAA ACT GCT CCA TTG CCC TAACTGGGTCTCACACCATC CAGACCCTCG 1437 Ala Glu Thr Ala Pro Leu Pro 395 CTAAGCTTAGAGGCCGCCAT CTACGTGGAA TCAGGTTGCT GTCAGGGTGT GTGGGAGGCT 1497 CTGGTTTCCTGAGAAACCAT CTGATCCTGC ATTCAAAGTC ATTCCTCTCT GGCTACTTCA 1557 CTCTGCACATGAGAGATGCT CAGACTGATC AAGACCAGAA GAAAGAAGAG ACTACCGGAC 1617 A 1618 398amino acids amino acid linear protein unknown 2 Met Asp Ser Ser Thr GlyPro Gly Asn Thr Ser Asp Cys Ser Asp Pro 1 5 10 15 Leu Ala Gln Ala SerCys Ser Pro Ala Pro Gly Ser Trp Leu Asn Leu 20 25 30 Ser His Val Asp GlyAsn Gln Ser Asp Pro Cys Gly Leu Asn Arg Thr 35 40 45 Gly Leu Gly Gly AsnAsp Ser Leu Cys Pro Gln Thr Gly Ser Pro Ser 50 55 60 Met Val Thr Ala IleThr Ile Met Ala Leu Tyr Ser Ile Val Cys Val 65 70 75 80 Val Gly Leu PheGly Asn Phe Leu Val Met Tyr Val Ile Val Arg Tyr 85 90 95 Thr Lys Met LysThr Ala Thr Asn Ile Tyr Ile Phe Asn Leu Ala Leu 100 105 110 Ala Asp AlaLeu Ala Thr Ser Thr Leu Pro Phe Gln Ser Val Asn Tyr 115 120 125 Leu MetGly Thr Trp Pro Phe Gly Thr Ile Leu Cys Lys Ile Val Ile 130 135 140 SerIle Asp Tyr Tyr Asn Met Phe Thr Ser Ile Phe Thr Leu Cys Thr 145 150 155160 Met Ser Val Asp Arg Tyr Ile Ala Val Cys His Pro Val Lys Ala Leu 165170 175 Asp Phe Arg Thr Pro Arg Asn Ala Lys Ile Val Asn Val Cys Asn Trp180 185 190 Ile Leu Ser Ser Ala Ile Gly Leu Pro Val Met Phe Met Ala ThrThr 195 200 205 Lys Tyr Arg Gln Gly Ser Ile Asp Cys Thr Leu Thr Phe SerHis Pro 210 215 220 Thr Trp Tyr Trp Glu Asn Leu Leu Lys Ile Cys Val PheIle Phe Ala 225 230 235 240 Phe Ile Met Pro Ile Leu Ile Ile Thr Val CysTyr Gly Leu Met Ile 245 250 255 Leu Arg Leu Lys Ser Val Arg Met Leu SerGly Ser Lys Glu Lys Asp 260 265 270 Arg Asn Leu Arg Arg Ile Thr Arg MetVal Leu Val Val Val Ala Val 275 280 285 Phe Ile Val Cys Trp Thr Pro IleHis Ile Tyr Val Ile Ile Lys Ala 290 295 300 Leu Ile Thr Ile Pro Glu ThrThr Phe Gln Thr Val Ser Trp His Phe 305 310 315 320 Cys Ile Ala Leu GlyTyr Thr Asn Ser Cys Leu Asn Pro Val Leu Tyr 325 330 335 Ala Phe Leu AspGlu Asn Phe Lys Arg Cys Phe Arg Glu Phe Cys Ile 340 345 350 Pro Thr SerSer Thr Ile Glu Gln Gln Asn Ser Thr Arg Val Arg Gln 355 360 365 Asn ThrArg Glu His Pro Ser Thr Ala Asn Thr Val Asp Arg Thr Asn 370 375 380 HisGln Leu Glu Asn Leu Glu Ala Glu Thr Ala Pro Leu Pro 385 390 395 1618base pairs nucleic acid single linear unknown CDS 339..1232 3 CGTGGAAGGGGGCTACAAGC AGAGGAGAAT ATCAGACGCT CAGACGTTCC CTTCTGCCTG 60 CCGCTCTTCTCTGGTTCCAC TAGGGCTGGT CCATGTAAGA ATCTGACGGA GCCTAGGGCA 120 GCTGTGAGAGGAAGAGGCTG GGGCGCGTGG AACCCGAAAA GTCTGAGTGC TCTCAGTTAC 180 AGCCTACCTAGTCCGCAGCA GGCCTTCAGC ACCATGGACA GCAGCACCGG CCCAGGGAAC 240 ACCAGCGACTGCTCAGACCC CTTAGCTCAG GCAAGTTGCT CCCCAGCACC TGGCTCCTGG 300 CTCAACTTGTCCCACGTTGA TGGCAACCAG TCCGATCC ATG CGG TCT GAA CCG 353 Met Arg Ser GluPro 1 5 CAC CGG GCT TGG CGG GAA CGA CAG CCT GTG CCC TCA GAC CGG CAG CCC401 His Arg Ala Trp Arg Glu Arg Gln Pro Val Pro Ser Asp Arg Gln Pro 1015 20 TTC CAT GGT CAC AGC CAT TAC CAT CAT GGC CCT CTA CTC TAT CGT GTG449 Phe His Gly His Ser His Tyr His His Gly Pro Leu Leu Tyr Arg Val 2530 35 TGT AGT GGG CCT CTT CGG AAA CTT CCT GGT CAT GTA TGT GAT TGT AAG497 Cys Ser Gly Pro Leu Arg Lys Leu Pro Gly His Val Cys Asp Cys Lys 4045 50 ATA CAC CAA AAT GAA GAC TGC CAC CAA CAT CTA CAT TTT CAA CCT TGC545 Ile His Gln Asn Glu Asp Cys His Gln His Leu His Phe Gln Pro Cys 5560 65 TCT GGC AGA CGC CTT AGC GAC CAG TAC ACT GCC CTT TCA GAG TGT CAA593 Ser Gly Arg Arg Leu Ser Asp Gln Tyr Thr Ala Leu Ser Glu Cys Gln 7075 80 85 CTA CCT GAT GGG AAC ATG GCC CTT CGG AAC CAT CCT CTG CAA GAT CGT641 Leu Pro Asp Gly Asn Met Ala Leu Arg Asn His Pro Leu Gln Asp Arg 9095 100 GAT CTC AAT AGA TTA CTA CAA CAT GTT CAC CAG CAT ATT CAC CCT CTG689 Asp Leu Asn Arg Leu Leu Gln His Val His Gln His Ile His Pro Leu 105110 115 CAC CAT GAG CGT GGA CCG CTA CAT TGC TGT CTG CCA CCC AGT CAA AGC737 His His Glu Arg Gly Pro Leu His Cys Cys Leu Pro Pro Ser Gln Ser 120125 130 CCT GGA TTT CCG TAC CCC CCG AAA TGC CAA AAT CGT CAA CGT CTG CAA785 Pro Gly Phe Pro Tyr Pro Pro Lys Cys Gln Asn Arg Gln Arg Leu Gln 135140 145 CTG GAT CCT CTC TTC TGC CAT CGG TCT GCC TGT AAT GTT CAT GGC AAC833 Leu Asp Pro Leu Phe Cys His Arg Ser Ala Cys Asn Val His Gly Asn 150155 160 165 CAC AAA ATA CAG GCA GGG GTC CAT AGA TTG CAC CCT CAC GTT CTCCCA 881 His Lys Ile Gln Ala Gly Val His Arg Leu His Pro His Val Leu Pro170 175 180 CCC AAC CTG GTA CTG GGA GAA CCT GCT CAA AAT CTG TGT CTT TATCTT 929 Pro Asn Leu Val Leu Gly Glu Pro Ala Gln Asn Leu Cys Leu Tyr Leu185 190 195 CGC TTT CAT CAT GCC GAT CCT CAT CAT CAC TGT GTG TTA CGG CCTGAT 977 Arg Phe His His Ala Asp Pro His His His Cys Val Leu Arg Pro Asp200 205 210 GAT CTT ACG ACT CAA GAG CGT TCG CAT GCT ATC GGG CTC CAA AGAAAA 1025 Asp Leu Thr Thr Gln Glu Arg Ser His Ala Ile Gly Leu Gln Arg Lys215 220 225 GGA CAG GAA TCT GCG CAG GAT CAC CCG GAT GGT GCT GGT GGT CGTGGC 1073 Gly Gln Glu Ser Ala Gln Asp His Pro Asp Gly Ala Gly Gly Arg Gly230 235 240 245 TGT ATT TAT CGT CTG CTG GAC CCC CAT CCA CAT CTA CGT CATCAT CAA 1121 Cys Ile Tyr Arg Leu Leu Asp Pro His Pro His Leu Arg His HisGln 250 255 260 AGC GCT GAT CAC GAT TCC AGA AAC CAC ATT TCA GAC CGT TTCCTG GCA 1169 Ser Ala Asp His Asp Ser Arg Asn His Ile Ser Asp Arg Phe LeuAla 265 270 275 CTT CTG CAT TGC TTT GGG TTA CAC GAA CAG CTG CCT GAA TCCAGT TCT 1217 Leu Leu His Cys Phe Gly Leu His Glu Gln Leu Pro Glu Ser SerSer 280 285 290 TTA CGC CTT CCT GGA TGAAAACTTC AAGCGATGCT TCAGAGAGTTCTGCATCCCA 1272 Leu Arg Leu Pro Gly 295 ACCTCGTCCA CGATCGAACA GCAAAACTCCACTCGAGTCC GTCAGAACAC TAGGGAACAT 1332 CCCTCCACGG CTAATACAGT GGATCGAACTAACCACCAGC TAGAAAATCT GGAGGCAGAA 1392 ACTGCTCCAT TGCCCTAACT GGGTCTCACACCATCCAGAC CCTCGCTAAG CTTAGAGGCC 1452 GCCATCTACG TGGAATCAGG TTGCTGTCAGGGTGTGTGGG AGGCTCTGGT TTCCTGAGAA 1512 ACCATCTGAT CCTGCATTCA AAGTCATTCCTCTCTGGCTA CTTCACTCTG CACATGAGAG 1572 ATGCTCAGAC TGATCAAGAC CAGAAGAAAGAAGAGACTAC CGGACA 1618 298 amino acids amino acid linear protein unknown4 Met Arg Ser Glu Pro His Arg Ala Trp Arg Glu Arg Gln Pro Val Pro 1 5 1015 Ser Asp Arg Gln Pro Phe His Gly His Ser His Tyr His His Gly Pro 20 2530 Leu Leu Tyr Arg Val Cys Ser Gly Pro Leu Arg Lys Leu Pro Gly His 35 4045 Val Cys Asp Cys Lys Ile His Gln Asn Glu Asp Cys His Gln His Leu 50 5560 His Phe Gln Pro Cys Ser Gly Arg Arg Leu Ser Asp Gln Tyr Thr Ala 65 7075 80 Leu Ser Glu Cys Gln Leu Pro Asp Gly Asn Met Ala Leu Arg Asn His 8590 95 Pro Leu Gln Asp Arg Asp Leu Asn Arg Leu Leu Gln His Val His Gln100 105 110 His Ile His Pro Leu His His Glu Arg Gly Pro Leu His Cys CysLeu 115 120 125 Pro Pro Ser Gln Ser Pro Gly Phe Pro Tyr Pro Pro Lys CysGln Asn 130 135 140 Arg Gln Arg Leu Gln Leu Asp Pro Leu Phe Cys His ArgSer Ala Cys 145 150 155 160 Asn Val His Gly Asn His Lys Ile Gln Ala GlyVal His Arg Leu His 165 170 175 Pro His Val Leu Pro Pro Asn Leu Val LeuGly Glu Pro Ala Gln Asn 180 185 190 Leu Cys Leu Tyr Leu Arg Phe His HisAla Asp Pro His His His Cys 195 200 205 Val Leu Arg Pro Asp Asp Leu ThrThr Gln Glu Arg Ser His Ala Ile 210 215 220 Gly Leu Gln Arg Lys Gly GlnGlu Ser Ala Gln Asp His Pro Asp Gly 225 230 235 240 Ala Gly Gly Arg GlyCys Ile Tyr Arg Leu Leu Asp Pro His Pro His 245 250 255 Leu Arg His HisGln Ser Ala Asp His Asp Ser Arg Asn His Ile Ser 260 265 270 Asp Arg PheLeu Ala Leu Leu His Cys Phe Gly Leu His Glu Gln Leu 275 280 285 Pro GluSer Ser Ser Leu Arg Leu Pro Gly 290 295 21 base pairs nucleic acidsingle linear unknown 5 ATCTTCACCC TCACCATGAT G 21 20 base pairs nucleicacid single linear unknown 6 CGGTCCTTCT CCTTGGAACC 20 372 amino acidsamino acid linear unknown 7 Met Glu Leu Val Pro Ser Ala Arg Ala Glu LeuGln Ser Ser Pro Leu 1 5 10 15 Val Asn Leu Ser Asp Ala Phe Pro Ser AlaPhe Pro Ser Ala Gly Ala 20 25 30 Asn Ala Leu Gly Ser Pro Gly Ala Arg SerAla Ser Met Leu Ala Leu 35 40 45 Ala Ile Ala Ile Thr Ala Leu Tyr Ser AlaVal Cys Ala Val Gly Leu 50 55 60 Leu Gly Asn Val Leu Val Met Phe Gly IleVal Arg Tyr Thr Lys Leu 65 70 75 80 Lys Thr Ala Thr Asn Ile Tyr Ile PheAsn Leu Ala Leu Ala Asp Ala 85 90 95 Leu Ala Thr Ser Thr Leu Pro Phe GlnSer Val Asn Tyr Leu Met Glu 100 105 110 Thr Trp Pro Phe Gly Glu Leu LeuCys Lys Ala Val Leu Ser Ile Asp 115 120 125 Tyr Tyr Asn Met Phe Thr SerIle Phe Thr Leu Thr Met Met Ser Val 130 135 140 Asp Arg Tyr Ile Ala ValCys His Pro Val Lys Ala Leu Asp Phe Arg 145 150 155 160 Thr Pro Ala LysAla Lys Leu Ile Asn Ile Cys Ile Trp Val Leu Ala 165 170 175 Ser Gly ValGly Val Pro Ile Met Val Met Ala Val Thr Gln Pro Arg 180 185 190 Asp GlyAla Val Val Cys Met Leu Gln Phe Pro Ser Pro Ser Trp Tyr 195 200 205 TrpAsp Thr Val Thr Lys Ile Cys Val Phe Ile Phe Ala Phe Val Val 210 215 220Pro Ile Leu Ile Ile Thr Val Cys Tyr Gly Leu Met Leu Leu Arg Leu 225 230235 240 Arg Ser Val Arg Leu Leu Ser Gly Ser Lys Glu Lys Asp Arg Ser Leu245 250 255 Arg Arg Ile Thr Arg Met Val Leu Val Val Val Gly Ala Phe ValVal 260 265 270 Cys Trp Ala Pro Ile His Ile Phe Val Ile Val Trp Thr LeuVal Asp 275 280 285 Ile Asn Arg Arg Asp Pro Leu Val Val Ala Ala Leu HisLeu Cys Ile 290 295 300 Ala Leu Gly Tyr Ala Asn Ser Ser Leu Asn Pro ValLeu Tyr Ala Phe 305 310 315 320 Leu Asp Glu Asn Phe Lys Arg Cys Phe ArgGln Leu Cys Arg Thr Pro 325 330 335 Cys Gly Arg Gln Glu Pro Gly Ser LeuArg Arg Pro Arg Gln Ala Thr 340 345 350 Thr Arg Glu Arg Val Thr Ala CysThr Pro Ser Asp Gly Pro Gly Gly 355 360 365 Gly Ala Ala Ala 370 391amino acids amino acid linear unknown 8 Met Phe Pro Asn Gly Thr Ala ProSer Pro Thr Ser Ser Pro Ser Ser 1 5 10 15 Ser Pro Gly Gly Cys Gly GluGly Leu Cys Ser Arg Gly Pro Gly Ser 20 25 30 Gly Ala Ala Asp Gly Met GluGlu Pro Gly Arg Asn Leu Ser Gln Asn 35 40 45 Gly Thr Leu Ser Glu Gly GlnGly Ser Ala Ile Leu Ile Ser Phe Ile 50 55 60 Tyr Ser Val Val Cys Leu ValGly Leu Cys Gly Asn Ser Met Val Ile 65 70 75 80 Tyr Val Ile Leu Arg TyrAla Lys Met Lys Thr Ala Thr Asn Ile Tyr 85 90 95 Ile Leu Asn Leu Ala IleAla Asp Glu Leu Leu Met Leu Ser Val Pro 100 105 110 Phe Leu Val Thr SerThr Leu Leu Arg His Trp Pro Phe Gly Ala Leu 115 120 125 Leu Cys Arg LeuVal Leu Ser Val Asp Ala Tyr Asn Met Phe Thr Ser 130 135 140 Ile Tyr CysLeu Thr Val Leu Ser Val Asp Arg Tyr Val Ala Val Val 145 150 155 160 HisPro Ile Lys Ala Ala Arg Tyr Arg Arg Pro Thr Val Ala Lys Val 165 170 175Val Asn Leu Gly Val Trp Val Leu Ser Leu Leu Val Ile Leu Pro Ile 180 185190 Val Val Phe Ser Arg Thr Ala Ala Asn Ser Asp Gly Thr Val Ala Cys 195200 205 Asn Met Leu Met Pro Glu Pro Ala Gln Arg Trp Leu Val Gly Phe Val210 215 220 Leu Tyr Thr Phe Leu Met Gly Phe Leu Leu Pro Val Gly Ala IleCys 225 230 235 240 Leu Cys Tyr Val Leu Ile Ile Ala Lys Met Arg Met ValAla Leu Lys 245 250 255 Ala Gly Trp Gln Gln Arg Lys Arg Ser Glu Arg LysIle Thr Leu Met 260 265 270 Val Met Met Val Val Met Val Phe Val Ile CysTrp Met Pro Phe Tyr 275 280 285 Val Val Gln Leu Val Asn Val Phe Ala GluGln Asp Asp Ala Thr Val 290 295 300 Ser Gln Leu Ser Val Ile Leu Gly TyrAla Asn Ser Cys Ala Asn Pro 305 310 315 320 Ile Leu Tyr Gly Phe Leu SerAsp Asn Phe Lys Arg Ser Phe Gln Arg 325 330 335 Ile Leu Cys Leu Ser TrpMet Asp Asn Ala Ala Glu Glu Pro Val Asp 340 345 350 Tyr Tyr Ala Thr AlaLeu Lys Ser Arg Ala Tyr Ser Val Glu Asp Phe 355 360 365 Gln Pro Glu AsnLeu Glu Ser Gly Gly Val Phe Arg Asn Gly Thr Cys 370 375 380 Ala Ser ArgIle Ser Thr Leu 385 390 369 amino acids amino acid linear unknown 9 MetGlu Leu Thr Ser Glu Gln Phe Asn Gly Ser Gln Val Trp Ile Pro 1 5 10 15Ser Pro Phe Asp Leu Asn Gly Ser Leu Gly Pro Ser Asn Gly Ser Asn 20 25 30Gln Thr Glu Pro Tyr Tyr Asp Met Thr Ser Asn Ala Val Leu Thr Phe 35 40 45Ile Tyr Phe Val Val Cys Val Val Gly Leu Cys Gly Asn Thr Leu Val 50 55 60Ile Tyr Val Ile Leu Arg Tyr Ala Lys Met Lys Thr Ile Thr Asn Ile 65 70 7580 Tyr Ile Leu Asn Leu Ala Ile Ala Asp Glu Leu Phe Met Leu Gly Leu 85 9095 Pro Phe Leu Ala Met Gln Val Ala Leu Val His Trp Pro Phe Gly Lys 100105 110 Ala Ile Cys Arg Val Val Met Thr Val Asp Gly Ile Asn Gln Phe Thr115 120 125 Ser Ile Phe Cys Leu Thr Val Met Ser Ile Asp Arg Tyr Leu AlaVal 130 135 140 Val His Pro Ile Lys Ser Ala Lys Trp Arg Arg Pro Arg ThrAla Lys 145 150 155 160 Met Ile Asn Val Ala Val Trp Gly Val Ser Leu LeuVal Ile Leu Pro 165 170 175 Ile Met Ile Tyr Ala Gly Leu Arg Ser Asn GlnTrp Gly Arg Ser Ser 180 185 190 Cys Thr Ile Asn Trp Pro Gly Glu Ser GlyAla Trp Tyr Thr Gly Phe 195 200 205 Ile Ile Tyr Ala Phe Ile Leu Gly PheLeu Val Pro Leu Thr Ile Ile 210 215 220 Cys Leu Cys Tyr Leu Arg Ile IleIle Lys Val Lys Ser Ser Gly Ile 225 230 235 240 Arg Val Gly Ser Ser LysArg Lys Lys Ser Glu Lys Lys Val Thr Arg 245 250 255 Met Val Ser Ile ValVal Ala Val Phe Ile Phe Cys Trp Leu Pro Phe 260 265 270 Tyr Ile Phe AsnVal Ser Ser Val Ser Val Ala Ile Ser Pro Thr Pro 275 280 285 Ala Leu LysGly Met Phe Asp Phe Val Val Ile Leu Thr Tyr Ala Asn 290 295 300 Ser CysAla Asn Pro Ile Leu Tyr Ala Phe Leu Ser Asp Asn Phe Lys 305 310 315 320Lys Ser Phe Gln Asn Val Leu Cys Leu Val Lys Val Ser Gly Ala Glu 325 330335 Asp Gly Glu Arg Ser Asp Ser Lys Gln Asp Lys Ser Arg Leu Asn Glu 340345 350 Thr Thr Glu Thr Gln Arg Thr Leu Leu Asn Gly Asp Leu Gln Thr Ser355 360 365 Ile

What is claimed is:
 1. An isolated and purified polynucleotide thatencodes a mammalian mu opioid receptor polypeptide, said polypeptidecomprising an amino acid residue sequence of SEQ ID NO:2.
 2. Theisolated and purified polynucleotide of claim 1, wherein saidpolynucleotide is a DNA molecule.
 3. The isolated and purifiedpolynucleotide of claim 1, wherein said polynucleotide comprises thenucleotide base sequence of SEQ ID NO:1.
 4. An isolated and purifiedpolynucleotide comprising a base sequence that is identical orcomplementary to a segment of at least 35 contiguous bases of SEQ IDNO:1.
 5. An expression vector comprising a polynucleotide that encodes amammalian mu opioid receptor polypeptide, wherein said polynucleotidehas a sequence having at least 35 contiguous bases identical to SEQ IDNO:1 or its complement and said polynucleotide is capable of hybridizingto SEQ ID NO:1 or its complement under a hybridization conditioninvolving 0.02M-0.15M NaCl at a temperature of about 50° C. to about 70°C.
 6. A recombinant host cell transfected with a polynucleotide thatencodes a mammalian mu opioid receptor polypeptide, wherein saidpolynucleotide has a sequence having at least 35 contiguous basesidentical to SEQ ID NO:1 or its complement and said polynucleotide iscapable of hybridizing to SEQ ID NO:1 or its complement under ahybridization condition involving 0.02M-0.15M NaCl at a temperature ofabout 50° C. to about 70° C.
 7. A process of preparing a mammalian muopioid receptor polypeptide comprising (a) transfecting a cell with apolynucleotide that encodes a mammalian mu opioid receptor polypeptide,wherein said polynucleotide has a sequence having at least 35 contiguousbases identical to SEQ ID NO:1 or its complement and said polynucleotideis capable of hybridizing to SEQ ID NO:1 or its complement under ahybridization condition involving 0.02M-0.15M NaCl at a temperature ofabout 50° C. to about 70° C.; (b) maintaining the transformed host cellunder biological conditions sufficient for expression of thepolypeptide; and (c) recovering the receptor.
 8. The isolated andpurified polynucleotide of claim 4, further defined as comprising thebase sequence of SEQ ID NO:1.
 9. The isolated and purifiedpolynucleotide of claim 4, further defined as encoding a full lengthmammalian mu opioid receptor wherein said polynucleotide is capable ofhybridizing to SEQ ID NO:1 or its complement under a hybridizationcondition involving 0.02M-0.15M NaCl at a temperature of about 50° C. toabout 70° C.
 10. The isolated and purified polynucleotide of claim 4,further defined as encoding a human mu opioid receptor wherein saidpolynucleotide is capable of hybridizing to SEQ ID NO:1 or itscomplement under a hybridization condition involving 0.02M-0.15M NaCl ata temperature of about 50° C. to about 70° C.
 11. The isolated andpurified polynucleotide of claim 4, further defined as comprising a basesequence that is identical or complementary to a segment of at least 55contiguous bases of SEQ ID NO:1.
 12. The expression vector of claim 5,wherein said polynucleotide has a sequence in which at least 55contiguous bases identical to or complementary to at least 55 contiguousbases of SEQ ID NO:1.
 13. The recombinant host cell of claim 6, whereinthe polynucleotide that encodes a mammalian mu opioid receptorpolypeptide has at least 55 contiguous bases that are identical to orcomplementary to 55 contiguous bases of SEQ ID NO:1.
 14. The process ofclaim 7, wherein the polynucleotide that encodes a mammalian mu opioidreceptor polypeptide has a sequence in which at least 55 contiguousbases are identical to or complementary to 55 contiguous bases of SEQ IDNO:1.
 15. An expression vector comprising a polynucleotide comprising abase sequence that is identical or complementary to a segment of atleast 35 contiguous bases of SEQ ID NO:1.